Lysine lactoylation prm target panel, method of constructing and application thereof

Abstract

This invention belongs to the field of proteomics and targeted mass spectrometry analysis technology, and relates to a lysine lactylation PRM target panel, its construction method, and its application. The panel may include all or a subset of the targets listed in the corresponding tables in Tables 1 to 3; the specific composition of the panel can be determined without requiring a specific preparation source as a necessary condition for identifying its members. The panel is stored in a computer-readable format and can be converted or rescheduled to one or more PRM acquisition methods based on liquid chromatography conditions, instrument platform, and retention time calibration results. It is used for lactylation-targeted detection, quantitative analysis, and cross-sectional comparison of low-input biological samples, clinical tissue samples, immune cell samples, rare cell samples, and single-cell-derived samples.

Description

Technical Field

[0001] This invention belongs to the field of proteomics and targeted mass spectrometry analysis technology, and relates to lysine lactylated PRM target panels, their construction methods and applications, especially to independent and source-related initial lactylated PRM target panels, large-scale lactylated PRM target panels and compressed lactylated PRM target panels, as well as their applications in lactylation targeted detection and quantitative analysis of low-input biological samples, clinical tissues, immune cells and single-cell-derived samples. Background Technology

[0002] Lysine lactylation is a type of post-translational modification of proteins associated with cellular lactate metabolism, and it can occur in both histones and non-histone proteins. Previous studies have suggested its potential involvement in chromatin regulation, metabolism, and immune-related processes; however, the causal relationship between different proteins, sites, and biological conditions still needs to be verified using site-specific experiments. Unlike conventional proteomics assays that only reflect protein expression levels, lactylation analysis requires simultaneous identification of the target peptide, the location of the lactylation within the peptide, and the corresponding ionic signal; therefore, it has independent detection targets and technical requirements.

[0003] Lactylated peptides in biological samples are typically characterized by low abundance, low modification occupancy, dispersed site distribution, and significant variability between different samples. In unenriched whole protein hydrolysates, lactylated peptides are further subjected to competition and interference from numerous unmodified peptides, co-washing eliminators, and background noise. Especially in clinical tissue sections, tumor-infiltrating immune cells, rare cells, and single-cell derived samples, the limited protein input further amplifies the technical challenge of consistently detecting lactylation signals.

[0004] Current lactylation discovery studies typically rely on peptide enrichment using anti-lactylated lysine antibodies or other affinity materials. This approach is suitable for expanding the identification range of lactylated peptides in the early stages of discovery; however, the enrichment effect is affected by antibody specificity, epitope bias, batch variability, elution efficiency, and non-specific adsorption, and usually requires a relatively high initial protein amount. For low-input samples, repeated antibody enrichment can not only lead to sample loss but also increase operational steps, batch bias, and detection costs, making it difficult to directly use for stable comparisons of large-scale clinical samples or rare cell samples.

[0005] Data-dependent acquisition (DDA) can obtain discovery-type spectra of lactylated peptides, but the selection of low-abundance lactylated precursors for secondary fragmentation is random, and the same lactylation site may not be continuously identified in different samples or batches. While data-independent acquisition (DIA) can expand data coverage, high-quality reference information and rigorous peak group determination are still required for stable quantification when lactylated peptide content is extremely low, modified site isoforms coexist, or sample input is limited. Therefore, the discovery-type identification results obtained in this way cannot be directly equated to a repeatable lactylation targeting panel.

[0006] Parallel reaction monitoring (PRM) can acquire high-resolution product ion spectra around a predetermined lactylated precursor, potentially improving target selectivity and cross-sample detection consistency. However, existing lactylated PRM applications are mostly ad hoc acquisition methods established for a small number of pre-selected peptides by researchers, lacking large-scale lactylated PRM target panels with well-defined compositions, validated by actual PRM acquisition, and reusable. Candidate lists derived solely from DDA results or reference spectral libraries cannot demonstrate that each lactylated target will form identifiable and quantifiable chromatographic peaks on a practical PRM platform.

[0007] When the number of lactylated precursor candidates reaches thousands to tens of thousands, the capacity of the PRM method may become insufficient. The overlap of numerous precursors within adjacent retention time intervals can lead to an excessive number of targets being monitored in a single cycle, thereby compressing residence time, reducing the number of sampling points per chromatographic peak, and decreasing quantitative stability. Therefore, it is necessary to dynamically allocate candidate targets to multiple PRM acquisition methods based on the measured or calibrated retention time of the lactylated precursor, local target density, and instrument acquisition capabilities, and to use the actual acquisition results for correction and rescheduling.

[0008] Even if a lactylation target has been detected by PRM in high-input validation samples, it does not mean that it is suitable for direct use in low-input, clinical, or single-cell-derived samples. For these samples, further screening of the validated large-scale target set is required based on indicators such as peak area, signal-to-background ratio, product ion co-elution relationship, peak shape integrity, and repeatability coefficient of variation to reduce the occupation of limited acquisition capacity by low-quality and unstable targets.

[0009] Therefore, the problem that needs to be solved in the field of lactylation detection is not the general problem of post-translational modification detection, but how to transform lactylation reference spectra or lactylation enrichment DDA identification results into a well-defined set of candidate targets, how to confirm its feasibility through actual PRM acquisition, and how to further obtain a high-quality target set that can be stably detected in low-input samples without antibody enrichment in the test stage.

[0010] Based on the aforementioned technical issues, it is necessary to establish initial PRM target panels with clear source relationships, large-scale PRM target panels, and compressed PRM target panels: initial panels are formed from PRM data resources, the initial panels are transformed into validated large-scale panels by actual PRM acquisition, and then compressed panels are formed by screening from the large-scale panels according to low-input application requirements, thereby taking into account the coverage of PRM targets, actual detectability, and low-input quantitative stability. Summary of the Invention

[0011] This invention addresses the technical problems of low abundance of lactylated peptides, insufficient cross-sample reproducibility of discovery-based identification, limited capacity of large-scale PRM methods, and unsuitability for repeated antibody enrichment with low-input test samples. It proposes a three-tiered PRM target panel system based on the source of lactylated targets and actual detection performance. The system sequentially includes an initial lactylated PRM target panel, a large-scale lactylated PRM target panel, and a compressed lactylated PRM target panel.

[0012] This invention does not simply name any set of lactylation targets as a panel, but rather assigns three independent panels to perform different technical functions in the establishment of the lactylation PRM method, forming a traceable source chain: the initial panel is used to transform lactylation spectra or identification resources into schedulable candidate targets; the large-scale panel is used to confirm the feasibility and detectability of candidate targets on a practical PRM platform; and the compressed panel is used to further meet the requirements of low-input, clinical, and single-cell source samples for acquisition capacity, signal quality, and reproducibility based on the large-scale panel. The compressed panels or application sub-panels listed in Tables 1 to 3 can also be independently stored, retrieved, and implemented according to their specific composition, without requiring a specific preparation source as a necessary condition for identifying their specific composition.

[0013] The first stage involves creating an initial PRM target panel for lactylated peptides. The inventors extract modified peptide sequences, the location of the lactylation modification within the peptide, the precursor ion mass-to-charge ratio, precursor ion charge state, retention time, and corresponding spectral information from a lactylated peptide reference spectral library or a list of lactylated peptide search results. This information can be linked to one or more candidate protein accessions and their corresponding lactylation sites and / or protein grouping information, forming a set of candidate lactylated PRM targets that have not yet been retained based on actual PRM detection results. The technical objective of this stage is to retain as many candidate targets as possible that have evidence of lactylation and can be converted into PRM acquisition targets.

[0014] The initial PRM target panel for lactylation can be formed through two lactylation-specific data routes. First, a high-coverage lactylation reference spectral library is established using chemically induced or simulated lactylated samples, and an initial panel for standard input or ultra-low input scenarios is formed accordingly. Second, the initial panel is formed directly using the DDA identification results of lactylated peptides enriched with anti-lactylated lysine antibodies or other affinity materials. The latter enrichment step is used for front-end lactylation target discovery, rather than being a necessary step for PRM detection of the test sample.

[0015] The second stage involves the formation of a large-scale lactylated PRM target panel. The inventors allocated candidate precursors from the initial panel to one or more PRM acquisition methods based on retention time and local target density. Actual PRM acquisition was performed using validation samples, and candidate targets that lacked identifiable chromatographic peaks or failed to meet basic detection requirements under the given conditions were excluded through peak extraction and detectability confirmation. The retained targets collectively constitute a large-scale lactylated PRM target panel validated on a real-world platform.

[0016] Large-scale panels are not simply name replacements for the initial panels; rather, they are executable technical objects obtained after the initial lactylation information has been validated through actual PRM acquisition. Their members can be traced back to the lactylated peptides and sites in the initial panel and have verifiable evidence of PRM acquisition, extraction, and identification on a real mass spectrometry platform. Multiple independent PRM acquisition methods can collectively constitute the executable acquisition format of the same large-scale panel.

[0017] The third stage involves the formation of a compressed lactylated PRM target panel. The inventors utilize pre-screened samples that are identical to or comparable to the target application scenario to perform one or more rounds of PRM acquisition on a large-scale panel. Quality screening is then conducted based on indicators such as absolute peak area, signal-to-noise ratio or signal-to-background ratio, relative abundance, product ion time correlation, effective product ion count, peak shape integrity, and coefficient of variation. The high-quality targets retained after screening constitute the compressed lactylated PRM target panel.

[0018] Compressed panels maintain the source correspondence between their members and large-scale panels and initial panels, but their technical purpose is further limited: by reducing low-quality or low-reproducibility targets, the single-sampling load is reduced, and the limited PRM acquisition capacity is focused on lactylated precursors that can be stably extracted and quantified in low-input samples. Based on clinical tissues, CD8... + Different needs for T cells, ultra-low input, or single-cell source samples can also lead to the formation of application sub-panels with well-defined compositions.

[0019] "Lysine lactylation" refers to the post-translational modification formed by the lactylation of the amino group on the lysine side chain. The lactylated peptides described in this invention can be derived from natural biological samples or from in vitro simulated lactylated or chemically modified samples used to establish a lactylation reference spectral library.

[0020] The “Lactylated Peptide Reference Spectrum Library” refers to a data set containing various information, including the sequence of lactylated modified peptides, the position of the lactylation modification in the peptide, the mass-to-charge ratio of the precursor ion, the charge state of the precursor ion, the product ion spectrum, and chromatographic retention time information. It can also be associated with the accession of one or more candidate proteins and the lactylation sites and / or protein grouping information in their corresponding protein sequences.

[0021] The "Search Results List for Lactylated Peptides" refers to a collection of lactylated peptides obtained after database searching, spectral matching, or peptide identification of lactylation-related liquid chromatography-mass spectrometry data. Each entry contains at least the lactylated modified peptide sequence and the location of the lactylation modification within the peptide, and may include one or more candidate protein accessions and their corresponding lactylation sites and / or protein grouping information, precursor ions, product ions, retention times, identification confidence levels, and false discovery rates.

[0022] "Lactylation PRM target" refers to the target lactylation peptide member and one or more precursor ions acquired by the mass spectrometer for controlling lactylation PRM data acquisition. Panel member identification is determined by the lactylation modified peptide sequence and the position of the lactylation modification within the peptide. The precursor ion mass-to-charge ratio and precursor ion charge state are acquisition attributes corresponding to the member; they can be recorded as explicit fields or calculated or converted based on the modified peptide sequence, the mass increment of the lactylation modification, and preset charge state rules. The same member may correspond to different precursor ion charge states or calibrated mass-to-charge ratio records under different instruments or acquisition conditions. When generating the PRM acquisition method, the product ion can be selected based on the corresponding lactylation reference spectrum or actual PRM data.

[0023] Membership of lactylated PRM targets, as referred to in this specification, is based on the lactylated peptide sequence and the position of the lactylation modification within that peptide. The same lactylated peptide may map to one or more candidate proteins; candidate protein accessions and their corresponding lactylation sites and / or protein grouping information are considered associative annotations and are not necessary conditions for distinguishing panel members. For non-unique peptides, multiple candidate protein accessions and their corresponding protein sequence sites can be recorded in a one-to-one correspondence set or pair. Changes in database version, protein merging rules, or peptide-protein mapping results do not alter the membership of the lactylated PRM target. Peptides with the same lactylated peptide sequence but different positions within the peptide are considered different lactylated PRM target members.

[0024] "Precursor ion dynamic scheduling" refers to the process of allocating candidate precursors to one or more PRM acquisition methods based on the chromatographic retention time of the lactylated candidate precursors and the target density within different time intervals. This process can further consider acquisition time windows, minimum residence time, chromatographic peak width, number of sampling points per peak, and time load of each method.

[0025] The "PRM acquisition method" referred to in this specification is implemented using parallel reaction monitoring based on high-resolution mass spectrometry, including non-timed PRM and scheduled PRM; both selectively isolate predetermined precursor ions and acquire corresponding high-resolution product ion spectra. The core implementation of this invention does not rely on triggered PRM, targeted secondary mass spectrometry, or MRM / SRM as essential technical features.

[0026] "Quality screening" refers to one or more rounds of screening of targets in a large-scale lactylation PRM target panel using pre-screened samples or repeatedly collected data to retain precursor ions suitable for subsequent lactylation detection or quantitative analysis. For quantification, at least three effective product ions are preferably retained, and a coefficient of variation of no more than 30% can be used as a repeatability standard.

[0027] “Low-input samples” include, but are not limited to, micro-volume cell lysates, clinical tissue sections, biopsy samples, rare immune cell samples, and single-cell-derived samples with a protein input of no more than 200 ng. “Single-cell-derived samples” refer to samples that are directly derived from a single cell, or obtained from a small number of cells (e.g., 1 to 100 cells) through micro-lysis, enzymatic digestion, transfer, or separation processes; when the specific result originates from a single cell, it can be called a single-cell level sample.

[0028] The panel members listed in Tables 1 to 3 are identified based on the lactylated peptide sequence and the position of the lactylation modification within the peptide, and are also recorded or associated with the corresponding precursor ion mass-to-charge ratio, charge state, retention time window, and identifier. Candidate protein accessions and their corresponding lactylation sites and / or protein grouping information are used as protein mapping annotations; there may be one or more such annotations, but they are not necessary for distinguishing panel members. Adjustments to the number or composition of product ions, retention time or retention time window, precursor ion charge state, collision energy, precursor or product ion mass-to-charge ratio calibration, liquid chromatography conditions, mass spectrometry platform, retention time calibration strategy, software data format, database version, protein merging rules, or peptide-protein mapping relationships do not change the membership of the corresponding lactylated target or its corresponding membership in the tertiary panel source chain.

[0029] The term "all or a subset" of the targets recorded in any of Tables 1 to 3 as used in this invention refers to a situation where it is not necessary to retain every record in the table, but the lactylated peptide sequence and the position of the lactylation modification within the peptide are still used as the basis for determining the identity of target members. The candidate protein accession and the lactylation site and / or protein grouping information in the corresponding protein sequence are used as association annotations and are not necessary conditions for distinguishing panel members. In different embodiments, the subset may contain no less than 50, no less than 100, or no less than 500 lactylated PRM targets in the corresponding table, or contain no less than 30%, no less than 50%, no less than 70%, or no less than 90% of the lactylated PRM targets in the corresponding table. The limitations regarding quantity or proportion in the claims are further limitations on the scope of the subset and do not negate the implementation of the complete panels of Tables 1 to 3 and other subsets as specific constituent panels.

[0030] In a first aspect, the present invention provides a compressed lactylated PRM target panel. The panel is an independent, computer-readable set of targets, formed from a large-scale lactylated PRM target panel validated through actual PRM acquisition through one or more rounds of quality screening, and can be imported, converted, or rescheduled for one or more PRM acquisition methods. In addition to the compressed panel defined by the source chain described above, the present invention also uses the member combinations listed in Tables 1 to 3 as specific component panels that can be independently stored and retrieved.

[0031] Furthermore, the specific component panel includes the sub-panel after CV screening of lung cancer-related samples listed in Table 1, and the CD8 sub-panel listed in Table 2. + Sub-panels for T-cell sample CV screening and compressed panels for ultra-low input and single-cell source samples listed in Table 3; each panel may include all or a subset of the targets recorded in the corresponding table, and specific preparation source may not be a necessary condition for identifying its composition.

[0032] Secondly, this invention provides a large-scale lactylated PRM target panel. The large-scale panel comprises lactylated PRM targets retained after actual PRM data acquisition, peak extraction, and detectability verification, and can be independently stored, retrieved, and rescheduled. The large-scale panel can also be formed into a compressed lactylated PRM target panel through one or more rounds of quality screening. The formation process, number of members, and executable acquisition methods of the large-scale panels under standard input conditions and ultra-low input conditions are described in Examples 1 and 3, respectively; their complete intermediate member details are not listed item by item in the appendix. In some embodiments, the large-scale lactylated PRM target panel comprises no fewer than 15,000 lactylated PRM targets that have undergone actual PRM data acquisition, peak extraction, and detectability verification, and is allocated or rescheduled to no fewer than 10 independent PRM acquisition methods. Each panel member can be traced back to the candidate target in the corresponding initial PRM target panel based on the lactylated modified peptide sequence and the position of the lactylation modification in the peptide, and has an identifiable chromatographic peak confirmed by actual PRM data peak extraction. The same panel member can be associated with one or more candidate protein accessions and their corresponding lactylation sites and / or protein grouping information in the protein sequence. The association information is not a necessary condition for member tracing. The multiple independent PRM acquisition methods together constitute the executable acquisition format of the large-scale lactylated PRM target panel. The number of acquisition methods, method allocation, and adjustment of acquisition parameters do not affect the product attribute of this panel as an independent computer-readable target set.

[0033] Thirdly, the present invention provides an initial lactylated PRM target panel. The initial panel contains candidate lactylated PRM targets that have not yet been retained as members based on actual PRM detection results, and can be independently stored, retrieved, and dynamically scheduled; the initial panel is formed based on a lactylated peptide reference spectrum library or a list of lactylated peptide search results, and can be used to form a large-scale lactylated PRM target panel or directly generate a PRM acquisition method.

[0034] Fourthly, the present invention provides a method for constructing a compressed lactylated PRM target panel, including the generation of an initial lactylated panel, dynamic scheduling of candidate precursors, actual PRM acquisition and verification, large-scale panel confirmation, pre-screening sample detection, and one or more rounds of quality screening.

[0035] Fifthly, the present invention provides the application of the aforementioned initial lactylation panel, large-scale panel, or compressed panel in controlling mass spectrometry for lactylation PRM data acquisition, as well as its application in the detection, quantitative analysis, and cross-sectional comparative analysis of lactylation in low-input biological samples, clinical tissues, immune cells, rare cells, and single-cell-derived samples.

[0036] In a sixth aspect, the present invention provides a non-transitory computer-readable storage medium for storing the lactylated PRM target panel, and a lactylated PRM target data acquisition system comprising a panel storage module, a conversion or scheduling module, and a data acquisition control module.

[0037] The beneficial effects of this invention include: 1. This invention establishes a continuous source chain of initial panels, large-scale panels, and compressed panels around the detection of lactylation, so that lactylation candidate targets start from spectral or identification evidence, undergo actual PRM detectability verification and application-oriented quality screening, and form target panels with clear composition and traceability.

[0038] 2. When performing PRM detection on low-input test samples, enrichment with anti-lactylated lysine antibodies or other affinity materials is not a necessary step. This can reduce sample loss during enrichment, washing, and transfer of clinical tissue, rare immune cell, and single-cell-derived samples, and reduce detection bias introduced by antibody batch and epitope bias.

[0039] 3. By pre-completing target validation and quality screening in high-volume or representative pre-screened samples, the resulting compressed lactylated PRM target panel can be directly used for the detection of samples with a protein input of approximately 200 ng, ultra-low cell counts, or even single-cell sources, allowing limited samples to be primarily used for data acquisition of the target lactylated peptides.

[0040] 4. Targeting lung cancer tissue sections and tumor-invasive CD8 sequences + The T-cell-based lactylation application sub-panel enables batch detection and paired comparison of the same lactylation targets without requiring lactylation antibody enrichment in the clinical samples to be tested, thereby improving the consistency of target coverage among different clinical samples.

[0041] 5. By retaining lactylated precursors with intact peak shapes, reasonable product ion relationships, and satisfactory reproducibility, the compressed panel reduces the occupation of PRM cycle capacity by low-quality targets and improves the stability and comparability of lactylation quantification results under low input conditions.

[0042] 6. The initial panel can be derived from a high-coverage lactylation reference spectrum library established from chemically modified samples, or from the DDA identification results of lactylation-enriched samples, enabling the front-end lactylation discovery resources to be transformed into executable PRM target panels that do not require subsequent antibody enrichment in the assay stage.

[0043] 7. The panel can be rescheduled according to the liquid chromatography conditions, mass spectrometry platform and retention time calibration results, while maintaining the membership relationship defined by the lactylated modified peptide sequence and the position of the lactylation modification in the peptide; one or more candidate protein accessions and their corresponding protein sequence sites and / or protein grouping information are retained or updated as associated annotations, which can be easily called up repeatedly under different experimental batches, database versions and instrument conditions. Attached Figure Description

[0044] Figure 1 The results of establishing and validating a panel for lactylated PRM targets under standard input conditions are shown. Figure 1 a shows the process of establishing a reference spectral library from chemically modified lactylated samples, generating initial PRM targets, performing dynamic scheduling, implementing validation injections, and forming a large-scale panel, as well as the number of precursor ions and PRM methods at each stage. Figure 1 b shows the process of forming a compressed lactylated PRM target panel from a large-scale panel through quality screening, as well as the number of lactylated peptides, modification sites and corresponding proteins retained; Figure 1 c shows the coefficient of variation distribution of the compressed panel in repeated detections at a protein input of 200 ng; Figure 1 d shows a volcano plot of the lactylated peptides that differentiate between sodium lactate-treated and control HeLa cells.

[0045] Figure 2 This presentation shows the results of subpanel screening and application of lactylated PRM targets for clinical tissue and immune cell samples. Among them, Figure 2 a shows the use of H1299 cells and CD8 + The process of implementing the first round of detectability screening and the second round of repeatability screening for T cell pre-screening samples, as well as the number of precursors and PRM methods retained at each stage; Figure 2 b shows the distribution of the coefficient of variation of repeated tests of the target after screening at a protein input of 200 ng; Figure 2 c shows the lactylated peptide segment that differs between a non-small cell lung cancer tumor tissue section and a paired adjacent normal tissue section; Figure 2 d shows tumor infiltration of CD8 + T cells and paired adjacent CD8 cells + Differentially lactylated peptides between T cells.

[0046] Figure 3 This demonstrates the establishment and applicability validation of a panel for lactylated PRM targets in ultra-low input and single-cell derived samples. Figure 3 a shows the process and corresponding quantities for establishing a lactylation reference spectrum library for ultra-low input scenarios, generating an initial panel, performing actual PRM verification, forming a large-scale panel, and further compressing it; Figure 3b shows the changes in the total signal intensity of lactylated peptides under different cell input levels; Figure 3 c shows the proportion of lactylated peptides with a repeated detection CV of less than 30% under different cell input levels; Figure 3 d shows the reproducible distribution of lactylated peptides detected in single-cell-derived samples; Figure 3 e shows the quantitative signal of the representative lactylated peptide AEVEGK(lac)DLPEHAVLK at different cell loading levels; Figure 3 f shows the product ion extraction ion chromatograms of the representative peptides at single-cell and serial cell input levels.

[0047] Appendix Explanation Table 1. Panel of lactacylated PRM targets after CV screening of lung cancer-related samples.

[0048] Table 2 CD8 + T-cell sample CV screening followed by lactylated PRM target panel.

[0049] Table 3. Lactylated compressed PRM target panel for ultra-low input and single-cell source samples.

[0050] The panels listed in Tables 1 to 3 are all terminal compression panels or application sub-panels: Tables 1 and 2 are for lung cancer-related samples and CD8 samples, respectively. + T-cell samples, and application sub-panels formed after further screening; Table 3 shows compressed panels for ultra-low input and single-cell source samples. The initial panels and large-scale panels under standard input and ultra-low input conditions are described in Examples 1 and 3, respectively, detailing their formation process, number of members, identity fields, and executable acquisition methods, but are no longer listed item by item in a complete member list. Each initial panel, large-scale panel, compressed panel, and application sub-panel can be used in combination according to the source chain, or independently converted or scheduled to the corresponding PRM acquisition method according to the detection purpose. Detailed Implementation

[0051] The following detailed embodiments further illustrate the concept and technical effects of the present invention to provide a full understanding of its purpose, features, and effects. Unless otherwise specified, the methods described are conventional methods in the art, and the materials described are readily available from publicly available commercial sources. The illustrative embodiments of the present invention are for explaining the invention and do not constitute an undue limitation on the scope of protection.

[0052] The lactylated PRM technology objects involved in this invention include a list of lactylated peptide search results, an initial lactylated PRM target panel, a large-scale lactylated PRM target panel, a compressed lactylated PRM target panel, and an application sub-panel. These technologies correspond to different stages from lactylated data resources to executable PRM targeting data acquisition.

[0053] The initial PRM target panel for lactylated peptides is generated based on a reference spectral library of lactylated peptides or a list of search results for lactylated peptides, and includes candidate lactylated PRM targets for establishing PRM acquisition methods. The initial panel can be generated from a reference spectral library established from chemically modified samples, or directly from the DDA identification results of lactylated enriched samples.

[0054] A large-scale lactylated PRM target panel is formed from an initial panel through actual PRM data acquisition, peak extraction, and detectability verification. A large-scale panel can be assigned to multiple independent PRM acquisition methods; these multiple independent methods together constitute the executable acquisition form of the panel.

[0055] Compressed lactylated PRM target panels are formed from large-scale panels after one or more rounds of quality screening, and are a high-quality subset of the corresponding large-scale panels. Application sub-panels can also be formed by selecting targets from large-scale or compressed panels, depending on sample type, tissue origin, or detection purpose.

[0056] Tables 1 to 3 record three terminal compressed panels or application sub-panels that require complete member details, and these tables form part of this specification. The appendices retain the Assay, lactylated modified peptide, precursor ion mass-to-charge ratio, precursor ion charge state, retention time window, protein Accession, lactylation site, and Identifier. The "lactylated modified peptide" field characterizes the position of the lactylation modification within the peptide using modification markers; the precursor ion mass-to-charge ratio and charge state are acquisition attributes; the "protein Accession" and "lactylation site" fields are protein mapping annotations. For non-unique peptides, one or more candidate protein Accessions and their corresponding lactylation sites and / or protein grouping information can be recorded; multiple candidate protein Accessions and their corresponding protein sequence sites correspond one-to-one in the same order, or are recorded in a protein Accession-protein sequence site pairing format. The protein mapping annotations are not a necessary condition for distinguishing panel members. Multiple lactylation sites recorded in Site 1, Site 2, and Site 3 are combined and recorded in the same "lactylation site" field in the appendices. All tables have a Positive acquisition polarity and an MS2 Scan Range of m / z 200–1,500. Therefore, the Polarity and Scan Range fields are not listed repeatedly in each target record. The initial panel and large-scale panel under standard input and ultra-low input conditions are described in Examples 1 and 3, including their formation process, number of members, identity fields, and executable acquisition methods. Their complete member details are not listed in the appendix.

[0057] Under different liquid chromatography conditions, mass spectrometry platforms, or retention time calibration strategies, the panels listed in Tables 1 to 3 can be rescheduled to use PRM acquisition methods with different acquisition time windows, residence times, peak widths, number of sampling points, collision energies, precursor ion charge states, or product ion combinations. Precursor ion or product ion mass-to-charge ratio calibration and software format conversion can also be performed. As long as the lactylated peptide sequence and the position of the lactylation modification within the peptide remain consistent, changes in the above acquisition attributes and parameters, as well as adjustments to the database version, protein merging rules, or peptide-protein mapping relationships, will not alter the membership of the corresponding targets or the panel membership relationships.

[0058] The term "all or a subset" of the targets recorded in any of Tables 1 to 3 as used in this invention refers to a situation where it is not necessary to retain every record in the table, but the lactylated peptide sequence and the position of the lactylation modification within the peptide are still used as the basis for determining the identity of target members. The candidate protein accession and the lactylation site and / or protein grouping information in the corresponding protein sequence are used as association annotations and are not necessary conditions for distinguishing panel members. In different embodiments, the subset may contain no less than 50, no less than 100, or no less than 500 lactylated PRM targets in the corresponding table, or contain no less than 30%, no less than 50%, no less than 70%, or no less than 90% of the lactylated PRM targets in the corresponding table. The limitations regarding quantity or proportion in the claims are further limitations on the scope of the subset and do not negate the implementation of the complete panels of Tables 1 to 3 and other subsets as specific constituent panels.

[0059] The "PRM acquisition method" referred to in this specification is implemented using parallel reaction monitoring based on high-resolution mass spectrometry, including non-timed PRM and scheduled PRM; both selectively isolate predetermined precursor ions and acquire corresponding high-resolution product ion spectra. The core implementation of this invention does not rely on triggered PRM, targeted secondary mass spectrometry, or MRM / SRM as essential technical features.

[0060] The lactylation detection and quantification results obtained by this invention can be used for scientific research, drug evaluation, sample comparison, biomarker research, or to provide intermediate detection information for subsequent analysis. The method of this invention itself does not include steps for directly determining whether a subject has a disease, determining a disease state, or developing a treatment plan based on the detection results.

[0061] For low-input cell samples, in one embodiment, the induced cells can be first prepared into a single-cell suspension and then aliquoted according to a predetermined cell number. Specifically, HeLa cells treated with 100 mM sodium lactate for 48 hours can be digested with 0.25% trypsin-EDTA buffer at 37°C for about 4 minutes to obtain a single-cell suspension. After counting using an automated cell counter, the suspension is diluted according to a predetermined cell number gradient. The cells are then resuspended in a lysis buffer, which may contain 1% sodium deoxycholate (SDC), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA), and 100 mM Tris-HCl at pH 8.5. The sample can be homogenized after heating at 95°C for 5 minutes, and the supernatant can be collected by centrifugation at 14,000 g for 5 minutes. The supernatant is enzymatically hydrolyzed overnight at 37°C, and then formic acid is added to a final concentration of about 0.5% to terminate the reaction. After removing SDC by centrifugation at 16,000 g, the sample is desalted using a C18 pipette tip and stored at -80°C for later use.

[0062] For ultra-low input samples based on a single-cell sorting platform, in one embodiment, the cellenONE single-cell sorting system can be used to dispense cells into multi-well plates pre-loaded with a lysis / enzymatic digestion mixture. The lysis / enzymatic digestion mixture may contain 0.2% dodecyl maltodextrin (DDM), 100 mM triethylammonium bicarbonate (TEAB), and 20 ng / μL trypsin. Cells can be screened based on morphological parameters, such as cell diameter of approximately 15–35 μm and elongation not exceeding 1.8, and sorted into 384-well plates at 1–100 cells / well. After sorting, the samples can be incubated at 37°C and approximately 70% humidity for approximately 2 hours, then cooled to approximately 20°C, acidified with 0.1% trifluoroacetic acid (TFA), and a portion of the sample is used for LC-MS analysis.

[0063] For clinical tissue section samples, in one embodiment, tumor tissue and its paired adjacent normal tissue can be embedded in OCT embedding medium, and serial sections can be prepared using a cryostat. Thicker sections can be used for parallel validation experiments such as immunoblotting, while thinner sections can be used for proteomics or targeted mass spectrometry analysis. This applies to tissue section samples and isolated and purified tumor-infiltrating CD8+. + T-cell samples can be processed using the same or similar procedures as those for the low-input samples described above, including lysis, reduction, alkylation, enzymatic digestion, desalting, and LC-MS detection.

[0064] It should be understood that the above sample preparation process is only a preferred embodiment applicable to the low-input application scenario of the present invention. Those skilled in the art can make equivalent adjustments to the lysis system, enzymatic digestion system, desalting method and sample loading method according to the sample type, sample volume and detection platform, without departing from the essence of the present invention.

[0065] General instructions for obtaining immune cell samples In some embodiments of the present invention, the pre-screening sample or the sample to be tested may be derived from peripheral blood CD8. + T cells or tumor-infiltrating CD8 + T cells.

[0066] CD8 from peripheral blood + In one embodiment, T cells can be obtained by collecting peripheral blood from a healthy donor, separating peripheral blood mononuclear cells (PBMCs) through density gradient centrifugation, removing residual red blood cells through erythrocyte lysis, and further employing CD8+... + CD8+ enriched using a T-cell isolation kit for negative sorting or other suitable methods. + T cells. Freshly isolated CD8 cells. + T cells can be activated using anti-human CD3 antibodies and anti-human CD28 antibodies and cultured in a T cell expansion medium; the medium may further contain penicillin, streptomycin and recombinant human interleukin-2 (IL-2) to obtain primary immune cell samples suitable for quality screening or method validation.

[0067] For tumor-infiltrating CD8 + In one embodiment, fresh tumor tissue and paired adjacent normal tissue from a lung adenocarcinoma patient are taken, washed with ice-cold phosphate buffer, weighed, and mechanically minced into small pieces. The tissue is then digested in a culture medium containing collagenase IV and deoxyribonuclease I, for example, at 37°C with shaking for approximately 30 minutes. The digested tissue suspension is then filtered through a cell filter, centrifuged to collect cells, and residual red blood cells are removed by erythrocyte lysis. The resulting cell pellet can be further processed using CD8+, a method suitable for tumor-infiltrating lymphocytes. + T-cell magnetic bead sorting kits or magnetic bead sorting methods were used to enrich and obtain tumor-infiltrating CD8 cells. + T cell samples.

[0068] It should be understood that peripheral blood CD8 + T cells and tumor-infiltrating CD8 + The method of T cell isolation is not limited to the specific reagent systems or equipment platforms mentioned above, as long as CD8 can be obtained to meet the requirements of subsequent low-input proteomics or PRM detection. +T cell samples can all be used to implement this invention.

[0069] Example 1: Establishment and validation of large-scale PRM target panels and compressed PRM target panels based on lysine lactylation. 1.1 Chemically induced lactylated modified peptides In this embodiment, to obtain a reference spectral library for subsequent PRM method development, samples containing the target modified peptides were prepared using chemical modification, and a reference spectral library of the modified peptides was constructed by acquiring high-resolution LC-MS / MS data. Figure 1 a). It should be noted that the chemical modification library construction method described is only a preferred method for obtaining the reference spectral library, and the development and application of the PRM method of the present invention are not limited to this library construction method.

[0070] Human cervical cancer HeLa cells were harvested and lysed using lysis buffer containing 200 mM HEPES (pH 8.0) with 8 M urea. The supernatant was collected after centrifugation at 16,000 g for 10 minutes. Protein concentration was determined using the BCA method. The protein solution was diluted to a concentration of 5 μg / μL. 10 μL (50 μg of protein) of the lysis buffer was taken and 1 μL of 130 mM TCEP was added to a final concentration of 10 mM. The mixture was incubated at 56 °C for 30 minutes. Then, 1 μL of 260 mM CAA was added to a final concentration of 20 mM, and the mixture was incubated at room temperature for 20 minutes. A laboratory-synthesized lactyl-NHS compound was accurately weighed and prepared as a 130 mM stock solution using anhydrous DMSO. This stock solution was added to the protein lysis buffer to a final concentration of 10 mM. The reaction mixture was incubated at 25 °C with shaking at 400 rpm for 1.5 hours. Add 1 volume of methanol (13 μL) and 1 / 4 volume of chloroform (3.25 μL), mix well and vortex for 10 min. Centrifuge at 16,000 g for 10 min and discard the supernatant. Wash the precipitate with methanol. Redissolve the precipitate in 8 M urea / 200 mM HEPES (pH 8.0) solution. Digest with LysC (1:100, w / w) and trypsin (1:100, w / w) sequentially at 37 °C and 400 rpm for 16 h. Desalt the obtained peptides using C18 ZipTip, dry by vacuum centrifugation, and store at -80 °C for later use.

[0071] 1.2 DDA Data Acquisition: Acquisition of Spectral Data from a Proof-of-Concept Lactylation Reference Spectrum Library HPLC parameters: The HPLC system was used in Trap-elute mode for separation. A nano-level ultra-high performance liquid chromatography (nanoUHPLC) system was employed, first loaded onto a PepMap Neo capture column (300 μm × 5 mm, 5 μm), and then separated on an Aurora Elite CSI C18 column (75 μm × 150 mm, 1.7 μm, IonOpticks). The column oven temperature was set to 50 °C. The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was 0.1% formic acid acetonitrile solution. The gradient elution time was set to 30 minutes.

[0072] Mass spectrometry parameters: The Orbitrap Astral (Thermo Fisher Scientific) ultra-high resolution mass spectrometry platform was used for data acquisition in DDA mode. The first-stage mass spectrometry (MS1) was acquired in the Orbitrap analyzer with a resolution of 180,000 (FWHM, @200 m / z), a scan range of m / z 300 to 1,600, and an automatic gain control target (AGC target) of 300%. The candidate precursor ions with the highest intensity were selected for secondary fragmentation sequentially, with an isolation window of 1.6 Th, high-energy collisional dissociation (HCD) as the fragmentation mode, and a normalized collision energy (NCE) of 30%. The second-stage mass spectrometry (MS2) fragment ions were detected on the Astral analyzer with a scan range of m / z 100 to 2000 and an AGC target of 200%.

[0073] Data Acquisition: DDA data were collected from chemically induced lactylated modified peptides. Each sample was collected three times and the data were combined for data search.

[0074] Data Search: The obtained raw data was searched against the UniProt human protein database using the SEQUEST search engine built into Proteome Discoverer 3.1. Lysine lactylation was defined as a variable modification, cysteine ​​aminomethylation as a fixed modification, and methionine oxidation as a variable modification. Peptides were identified at a false discovery rate (FDR) of 1%. A proof-of-concept lactylation reference spectral library was constructed, including precursor ion m / z, secondary fragment ion spectra, chromatographic retention times, and modification site annotations. In this embodiment, the reference spectral library is named ChemIntelligence Klac Library, containing 22,388 unique lactylated peptides, corresponding to 19,413 lactylation modification sites on 2,826 proteins, for subsequent development of PRM methods. Figure 1 a).

[0075] 1.3 Import the reference spectrum library into the targeted mass spectrometry analysis software and generate a PRM target list. A PRM target list was generated using targeted mass spectrometry analysis software. In this embodiment, Skyline (v24.1.1.398) was used. The parameter settings are as follows: Peptide parameters: set the protease to Trypsin [KR / P], allowing a maximum of 3 missed cleavage sites; set the peptide length range to 6 to 45 amino acids; enable the "Auto-select all matching peptides" option.

[0076] Modification parameters: The fixed modification is set to carbamidomethylation of cysteine; the variable modification is set to lactylation of lysine (+72.0211 Da) and oxidation of methionine, with a maximum of 3 variable modifications allowed and a maximum of 3 neutral loss.

[0077] Product ion parameters: precursor ion charge states are set to +2 and +3; product ion types are b ions and y ions; product ion charge states are set to +1 and +2; product ion matching tolerance is set to 20 ppm; the full scan is set using DDA parameters: MS1 screening is performed using the Orbitrap instrument with a resolution of 180,000@200 m / z, and MS / MS screening is performed using 20 ppm; the retention time window is set to 2 minutes, and the measured value is used first when the actual retention time is available.

[0078] Product ion screening: 15 product ions were selected for each precursor, and the library matching tolerance was set to 0.5 Da.

[0079] Data Import: After completing the above parameter configuration, export the DDA data search results corresponding to the proof-of-concept lactylation reference spectrum library in pepXML format, screen for peptides with a Percolator q-value < 0.01, and import them into the targeted mass spectrometry analysis software (Skyline v24.1.1.398 in this embodiment) to construct the reference library (BiblioSpec, i.e., the .blib file in this embodiment). Perform peak shape extraction on all candidate precursors, exclude precursor ions with unidentifiable chromatographic peaks, and obtain a list of effective candidate precursors for subsequent dynamic scheduling of precursor ions.

[0080] 1.4 Generation of precursor ions through dynamic scheduling and PRM acquisition method The aforementioned reference library file is invoked, and the precursor ion dynamic scheduling module is used to dynamically schedule all valid precursors in the standard dosage lactylation reference spectrum library. In this embodiment, the precursor ion dynamic scheduling module is implemented using PRMConductor (v1.0.4344).

[0081] The parameters are set as follows: Select "Keep All Precs"; set the acquisition time window for each precursor to 1.4 minutes; set the minimum residence time to 10 ms; set the peak width to 10 seconds; ensure at least 7 acquisition points for each peak; retain only a single charge state for each precursor.

[0082] Following the above scheduling, 17,543 precursor ions from the proof-of-concept lactylation reference spectral library were assigned to 18 independent PRM methods, each of which can be imported into the mass spectrometer control software. Figure 1 a) Since these methods are generated based on computer simulation scheduling of the DDA spectral library, they need to be validated and calibrated through actual PRM acquisition. The 17,543 candidate precursors together constitute the initial PRM target panel for standard input lactylation, and this candidate set can be used to form a PRM-validated large-scale PRM target panel for standard input lactylation.

[0083] 1.5 PRM Data Acquisition: Validation Injection and Sample Detection Liquid chromatography parameters: The liquid chromatography method used for PRM acquisition is completely consistent with that used for DDA acquisition. Mass spectrometry parameters: PRM data acquisition was performed using a Stellar mass spectrometer. Adaptive Retention Time (RT) was enabled. Under different sample matrices, processing methods, or formulations, it is preferable to first generate an rtbin file for retention time calibration and then use this rtbin file in subsequent relative quantification data acquisitions.

[0084] MS1 scans were performed in the m / z range of 300–1,600 at a scan rate of 33 kDa / s. Maximum Injection Time was set to custom mode, corresponding to 10 ms. Automatic Gain Control (AGC) was set to standard mode, with an absolute AGC target of 3 × 10⁻⁶. 4 .

[0085] MS2 scanning used HCD for fragmentation, with NCE set to 30%. The MS2 scan range was 200–1,500 m / z, and the scan rate was 125 kDa / s. The AGC target was set to standard, and the absolute AGC target was 1×10⁻⁶. 4The maximum ion implantation time is set to dynamic mode. Loop control is set to "All".

[0086] Data acquisition: Chemically induced lactylated peptide samples (i.e., HeLa samples chemically modified with lactyl-NHS, the same as those used in library construction) were individually validated by single injections using 18 newly generated PRM methods on a Stellar mass spectrometer. The initial PRM acquisition methods generated based on the DDA reference spectral library were validated and calibrated on the actual platform.

[0087] 1.6 PRM Data Import and Large-Scale Panel Validation of PRM Targets for Lactylation The raw data acquired by PRM are imported into the targeted mass spectrometry analysis software for peak extraction. The software parameters in this stage remain consistent with those in the target list generation stage, and are adjusted appropriately based on the characteristics of the PRM data, as follows: Peptide and modification parameters: The same peptide and modification parameters as in step 1.2 are used, i.e. the protease is set to Trypsin [KR / P], and a maximum of 3 missed cleavage sites are allowed; the fixed modification is cysteine ​​aminomethylation, and the variable modification is lysine lactylation and methionine oxidation.

[0088] Product ion parameter adjustments: Precursor ion charge states are set to +2 and +3; product ion types are b ions and y ions; product ion charge states are set to +1 and +2; product ion quantity is set to retain 15 product ions per precursor; ion matching tolerance is adjusted from 20 ppm in the DDA stage to 0.5 Da (to adapt to the actual resolution requirements of Orbitrap detection in PRM acquisition); full scan settings are switched to PRM mode: MS1 screening is disabled, MS2 screening is set to PRM mode, matching tolerance is set to 0.5 Da; retention time window is set to 2 minutes.

[0089] Data import and screening: After importing the raw data obtained from PRM into the targeted mass spectrometry analysis software to complete peak extraction, precursor ions with no identifiable chromatographic peaks are first excluded, and then the precursors are initially screened according to the preset quality standards. For precursors that pass the screening, the method allocation is further re-utilized using the precursor ion dynamic scheduling module to generate an optimized PRM method suitable for the actual detection platform.

[0090] Parameter settings: Select "Keep All Precs"; set the acquisition time window for each precursor to 1.4 minutes; set the minimum residence time to 10 ms; set the peak width to 9 seconds; ensure at least 8 acquisition points per peak; retain only a single charge state for each precursor. After a single validation injection and peak extraction screening, the detectable precursors were reassigned, and the proof-of-concept lactylated ChemIntelligence Library ultimately identified 17,494 precursor ions, which were reassigned to 21 PRM methods (…). Figure 1 a).

[0091] 1.7 Formation of large-scale emulsified PRM target panels Following actual PRM data acquisition, peak extraction, and detectability confirmation, a total of 17,494 lactylated precursor ions were identified. These 17,494 precursor ions, validated through actual PRM acquisition, collectively constitute a large-scale lactylated PRM target panel and were reassigned to 21 independent PRM acquisition methods. These 21 independent PRM acquisition methods together constitute the executable acquisition format for this large-scale lactylated PRM target panel, enabling control of the mass spectrometer to acquire PRM data from the 17,494 lactylated precursor ions.

[0092] The large-scale lactylated PRM target panel consists of the aforementioned 17,494 precursor ions verified by actual PRM acquisition. Its formation process, number of members, and executable acquisition methods are described in this embodiment. To keep the length of the specification, the complete list of intermediate members will not be listed item by item in the appendix.

[0093] 1.8 Method Application Validation: Dynamic Quantification of Lactylation in HeLa Cells under Lactate Stimulation PRM detection and first-round quality screening: HeLa cell positive samples treated with sodium lactate (final concentration 100 mM) for 48 hours were used as pre-screening samples. The large-scale lactylated PRM target panel formed in step 1.7 was used as the screening target. The above 21 PRM methods were run on a Stellar mass spectrometer.

[0094] Data Import and Screening: The collected raw data was imported into Skyline. Peak shape identification was performed according to the parameters of the PRM data import and peak extraction stage in Example 1.6, and precursors with no identifiable chromatographic peaks were excluded. The quality screening results were evaluated using the following criteria: absolute peak area > 500; signal-to-background ratio (S / B) > 3; relative abundance > 0.05; time correlation > 0.8; number of effective product ions ≥ 3.

[0095] Parameter settings: Based on this, PRM Conductor rescheduled these precursors, setting the acquisition time window to 1.4 minutes, the minimum residence time to 10 ms, the peak width to 10 seconds, and at least 7 acquisition points per peak. A balanced load mode was used for compression, resulting in a compressed PRM target panel containing 1,122 lactylated peptides for subsequent quantitative analysis. Figure 1 (b) The 1,122 lactylated peptides together constitute a compressed lactylated PRM target panel, and belong to a subset of the standard dosage large-scale lactylated PRM target panel described in step 1.7.

[0096] Quantitative analysis: HeLa cells were treated with sodium lactate (L-lactate, final concentration 100 mM) for 48 hours as a positive stimulation group, and untreated cells were used as a control group. Each group was a triple biological replicate, and the protein input per replicate was 200 ng (this amount is only an example, and the present invention is also applicable to lower or slightly higher protein input amounts. The 200 ng amount demonstrates its effectiveness in low input scenarios). Peptides were prepared according to the standard trypsin digestion process, purified by C18 desalting column, and data were collected using a compressed PRM target panel finally established in Example 1.

[0097] Maintaining the PRM settings in Skyline, the raw mass spectrometry data were imported into Skyline, peak areas were extracted, and normalization was performed using total ion current (TIC). Records of peptides that were stably detected across all paired samples, had no truncation peaks, and were uniquely gene-specific peptides were further selected only when peptide signals were assigned to specific genes or proteins and downstream differential statistics were performed; this unique peptide limitation was not used as a constraint on the composition or membership of the compressed PRM target panel. For differentially lactylated peptides in cell samples, statistical analysis was performed using a two-tailed Student t-test combined with Benjamini-Hochberg correction.

[0098] Repeated assays on sodium lactate-treated HeLa samples showed that 762 out of 1,122 peptides targeted by the compressed PRM target panel exhibited excellent analytical reproducibility (CV < 30%), demonstrating the high precision and reliability of the method. Figure 1 c).

[0099] Quantitative analysis showed that, compared with the control group, 48 lactylated peptides underwent significant changes after lactate treatment (45 upregulated and 3 downregulated, with fold changes >2.0 or <0.5, and corrected P<0.05). Figure 1 d).

[0100] This embodiment demonstrates that for amplifiable cell line samples, a compressed lactylated PRM target panel can be further developed from a large-scale lactylated PRM target panel through actual PRM acquisition verification and single-round quality screening. The compressed lactylated PRM target panel is defined by a clearly defined lactylated modified peptide sequence, the position of the lactylation modification within the peptide, the precursor ion mass-to-charge ratio, the precursor ion charge state, product ion information, and retention time information. It can also be annotated with one or more candidate protein accessions and their corresponding lactylation sites and / or protein grouping information. This panel can be imported, converted, or rescheduled as a computer-readable target set for PRM acquisition methods and used to control a mass spectrometer to perform lactylation-targeted data acquisition for low-input samples. This panel achieves high reproducibility and high-throughput detection at a protein input of 200 ng, demonstrating its practicality as a reusable PRM target panel.

[0101] Example 2: Lactylated PRM target subpanel for clinical and immune cell samples 2.1 Method Design In actual clinical research, protein materials derived from tumor tissue sections or immune cells in the tumor microenvironment are usually very limited, and conventional antibody enrichment strategies often require high protein input, making them unsuitable for low-input clinical samples. To address these issues, this invention constructs a targeted PRM detection strategy based on a reference spectral library. By combining a first-round quality screening of amplifiable cells with a second-round quality screening of clinical samples, high-throughput and stable detection of lactylation modifications can be achieved under extremely low sample input conditions.

[0102] The core idea of ​​this strategy is as follows: First, representative cell line samples are used to perform PRM validation on candidate precursor ions in the reference spectral library to screen for candidate peptides with stable signal responses in biological samples. Then, a precursor ion dynamic scheduling module is used to reschedule the candidate precursors and construct an optimized PRM method. Finally, one or more rounds of quality screening are used to obtain a high-quality target list suitable for clinical samples. In this embodiment, two rounds of screening are preferably used as an example for illustration; the reference spectral library used is named ChemIntelligence Klac Library; the precursor ion dynamic scheduling module is implemented through PRM Conductor.

[0103] 2.2 First round of screening: based on expandable cells The H1299 human non-small cell lung cancer cell line was used as the representative cell line for the first round of pre-quality screening of clinical lung cancer tissue samples. After inducing H1299 cells with 80 mM sodium lactate for 48 hours, a simulated lactylated digest of H1299 cells was obtained. After enzymatic desalting according to the standard procedure, the full-library PRM method (21 methods, covering 17,494 precursors) established in Example 1 was used for PRM detection on Stellar. The protein input was 200 ng (as an example of a low-input sample).

[0104] Data Import and Screening: Raw data was imported into Skyline and peak shape identification was performed using the same parameters as PRM, excluding precursors with no identifiable chromatographic peaks. PRM Conductor was run to evaluate the pre-screening results using the following criteria: absolute peak area > 500; signal-to-background ratio (S / B) > 3; relative abundance > 0.05; time correlation > 0.8; effective product ion count ≥ 3. After the first round of screening, a total of 1,628 precursors meeting the above criteria were found in H1299 cells. Figure 2 a). The 1,628 precursors can serve as a candidate set for subsequent CV screening.

[0105] Parameter settings: Based on this, PRM Conductor rescheduled 1,628 precursors, set the peak width to 9 seconds, the residence time to 10 ms, at least 8 acquisition points per peak, and the acquisition window to 1.4 minutes, and assigned them to 3 independent PRM methods as detection methods for the subsequent second round of screening.

[0106] For primary immune cell pre-screening samples, in one embodiment, peripheral blood from healthy donors is collected, and PBMCs are obtained by density gradient centrifugation, followed by further separation and purification of CD8. + T cells were activated and expanded in vitro using an anti-CD3 / anti-CD28 stimulation system and a culture system containing IL-2. Subsequently, the peripheral blood-derived CD8 cells were treated with 40 mM sodium lactate. + T cells, after 48 hours of enzymatic desalting according to standard procedures, were used as representative primary immune cell samples for pre-quality screening. Following the same PRM detection and quality screening process as the H1299 samples described above, 2,050 valid precursors were obtained and allocated to three PRM methods, serving as the basis for subsequent clinical immune cell sample screening and detection. Figure 2 a). The aforementioned 2,050 effective precursors can be used as subsequent CD8. + Candidate set for T cell sample CV screening.

[0107] 2.3 Second round of screening: Screening based on technical repeatability and reproducibility A second round of testing was performed on tumor cell line samples using a triple-technology repeat method to screen for precursor ions with a CV < 30%.

[0108] For samples from the H1299 source, the raw data obtained from three technical repetitions were imported into Skyline, peak shape identification was performed using the same parameters, and the following two quality control criteria were added: (1) Precursors with a CV > 30% in three technical replicates were excluded; (2) Exclude precursors that have truncated peaks.

[0109] In the second round of screening, the parameters of the precursor ion dynamic scheduling module can be further adjusted to: minimum residence time of 15 ms, peak width of 8 seconds, at least 7 collection points per peak, and a collection window of 1.4 minutes. After the second round of fine screening, 1,255 high-quality, quantifiable precursors without truncation peaks were ultimately retained from the H1299-derived clinical samples and allocated to 3 final clinical PRM methods, which can be used for batch detection of large cohorts of clinical samples. Figure 2 (ab). The 1,255 high-quality, quantifiable precursors selected and retained constitute a sub-panel of lactylated PRM targets suitable for lung cancer tissue sample detection, the specific composition of which is described in Table 1.

[0110] For CD8 + For T cell samples, the parameters of the precursor ion dynamic scheduling module can be adjusted as follows: minimum residence time 12ms, peak width 8 seconds, at least 7 collection points per peak, and collection window 1.4 minutes. Using a balanced loading mode, a compressed PRM target panel was finally obtained, containing 771 quantifiable precursors without truncation peaks. Figure 2 ab). The 771 high-quality, quantifiable precursors selected and retained together constitute the basis for CD8. + The specific composition of the lactylated PRM target sub-panel for T cell sample detection is described in Table 2.

[0111] 2.4 Detection of PRM for Lactylation in Clinical Samples Using the PRM method obtained from the above screening, clinical lung cancer tissue, adjacent normal tissue, and tumor-infiltrating CD8 were analyzed. + T cells and adjacent CD8 cells + T cell samples were subjected to lactylation detection.

[0112] Sample preparation: In one embodiment, paired tumor tissue and adjacent normal tissue from three patients with non-small-cell lung cancer (NSCLC) were taken, embedded in optimal cutting temperature (OCT) embedding medium, and serial tissue sections were prepared using a cryostat.

[0113] In one implementation, tumor-infiltrating CD8 + T cells are derived from fresh tumor tissue and paired adjacent normal tissue. Specifically, tissue samples are washed with ice-cold PBS, minced, and then enzymatically digested at 37°C in a medium containing collagenase IV and deoxyribonuclease I. The digested cell suspension is filtered, centrifuged, and residual red blood cells are removed before further processing using CD8+. + T-cell magnetic bead sorting method enriches tumor-infiltrating CD8 cells. + T cells were used for subsequent low-input sample preparation and PRM detection.

[0114] 10 μm thick tissue sections and purified tumor-infiltrating CD8 + T cell samples are preferably processed using the same or similar preparation procedures as low-input samples.

[0115] For low-input tissue section samples and CD8 + In one embodiment, T cell samples were added to a lysis buffer at pH 8.5, the lysis buffer containing 1% SDC, 10 mM TCEP, 40 mM CAA, and 100 mM Tris-HCl. The samples were homogenized after heating at 95°C for 5 minutes and centrifuged at 14,000 g for 5 minutes to remove insoluble matter. The supernatant was collected and enzymatically digested overnight at 37°C. After digestion, formic acid was added to a final concentration of approximately 0.5% to terminate the reaction, and the samples were centrifuged at 16,000 g to remove SDC. The resulting supernatant was desalted using a C18 electrolyte and used for subsequent PRM assays.

[0116] In some implementations, to ensure consistency in testing between different clinical samples, tissue section samples and CD8... +The protein input for T-cell samples was controlled at approximately 200 ng per injection. Raw mass spectrometry data were imported into Skyline, peak area was extracted, and TIC normalization was performed. Only when peptide signals were assigned to specific genes or proteins and downstream differential statistics were performed, records of peptides that were stably detected in paired samples, had no truncated peaks, and were uniquely gene-specific were further selected; this unique peptide restriction was not used as a limitation on panel composition or membership as described in Tables 1 or 2. For differential analysis between tumor and adjacent normal samples, a two-tailed paired t-test was performed on the log2-transformed tumor / adjacent normal ratio; when the fold change in tumor / adjacent normal ratio was >2 or <0.5 and P <0.05, it was considered a candidate for significantly changed lactylated peptides.

[0117] Quantitative analysis: Lactylation analysis of frozen tissue sections from non-small cell lung cancer (NSCLC) showed that targeted quantitative analysis of three pairs of paired NSCLC tumor and adjacent normal tissue sections stably quantified 890 unique lactylated peptides across 593 proteins, containing 1,115 modification sites. Of these, 163 peptides showed significantly upregulated lactylation levels, while 7 peptides showed significantly decreased lactylation levels. Figure 2 c).

[0118] Targeting tumor-infiltrating CD8 + Lactylation analysis of T cells showed that, through the analysis of three pairs of paired NSCLC tumors and adjacent invasive CD8 cells, + Targeted quantitative analysis of T cells stably quantified 476 unique lactylated peptides across 588 proteins, containing 1,141 modification sites. Among these, 28 peptides showed significantly upregulated lactylation levels, corresponding to 31 modification sites on these 28 proteins. Figure 2 d).

[0119] 2.5 Summary of Methods This embodiment demonstrates that, for clinical tissue sections and rare immune cell samples with extremely limited protein input, a two-round quality screening process can be used to generate a lactylated PRM target sub-panel suitable for specific sample types from a large-scale lactylated PRM target panel. The first round of screening uses expandable cell lines or expanded primary cells as pre-screening samples to identify candidate precursors with reliable signals in biological samples. The second round of screening uses repeated acquisition techniques, with CV not exceeding 30% and peak integrity as criteria, to further screen for high-quality precursors with high reproducibility. The resulting lactylated PRM target sub-panel for lung cancer tissue samples and CD8... +The T-cell sample lactylation PRM target sub-panels all have clearly defined target compositions and can be converted or rescheduled into one or more executable PRM acquisition methods to control the mass spectrometer for lactylation-targeted data acquisition of low-input clinical samples or immune cell samples. This embodiment illustrates that the PRM target panels can be further developed into application sub-panels with specific uses based on sample type and application scenario.

[0120] Example 3: Lactylated PRM target panel suitable for ultra-low input and single-cell derived samples 3.1 Establishment of the ChemIntelligence Library with Ultra-Low Input Liquid chromatography parameters for DDA data acquisition in ultra-low input samples: To support targeted detection of emulsification in ultra-low input samples, the data acquisition method during the reference spectral library construction phase was optimized. Liquid chromatography was performed in direct injection mode, i.e., without a trapping column; other settings were essentially the same as conventional DDA settings.

[0121] Mass spectrometry parameters: The Orbitrap Astral Zoom mass spectrometer is connected to the FAIMS Pro module, and the compensation voltage (CV) is set to... 48 V. DDA acquisition mode is used; except for the settings described above, other parameters are consistent with the DDA parameters described in step 1.2 of Example 1. Pre-accumulation mode is enabled.

[0122] Data acquisition: DDA data acquisition was performed on the above platform using the same chemically modified lactylated samples as in Example 1 to construct a lactylated reference spectrum library optimized for ultra-low input scenarios.

[0123] Data search: Similar to Example 1, the SEQUEST engine was used for searching. The final result was a lactylation reference spectral library suitable for ultra-low-input scenarios; in this embodiment, this reference spectral library is named the ultra-low-input ChemIntelligence Klac Library, containing 26,835 lactylated peptides, corresponding to 24,588 lactylation sites of 3,721 proteins. Figure 3 a).

[0124] 3.2 Establishing a PRM Method for Ultra-Low Input Scenarios Based on the ultra-low input lactylation ChemIntelligence Library, the spectral library was imported according to the Skyline parameters described in Example 1 to generate a PRM target list. The target list was imported into PRM Conductor, and precursor ions were assigned according to the following parameters: minimum residence time 10 ms; peak width 10 seconds; at least 7 acquisition points per peak; acquisition window 1.4 minutes; each precursor retaining only a single charge state. After scheduling, 18,157 precursors from the low-input lactylation reference spectral library were assigned to 15 independent PRM methods. These 18,157 candidate precursors can be used to form a PRM-validated ultra-low input large-scale lactylation PRM target panel.

[0125] 3.3 PRM Method Confirmation and Reallocation Subsequently, single-shot validation injections were performed on these PRM methods using lactyl-NHS-modified HeLa lysis buffer on a Stellar mass spectrometer, and peak extraction was performed using Skyline. After excluding precursors with unidentifiable peaks under these validation conditions, the target list was imported into PRM Conductor for reassignment; ultimately, 18,012 precursors yielded identifiable peaks in this validation data and were reassigned to 19 PRM methods. Figure 3 a) The 18,012 precursors together constitute a large-scale emulsified PRM target panel suitable for ultra-low input scenarios; the 19 PRM acquisition methods together constitute its executable acquisition form. To keep the length of the specification, the complete list of intermediate members will not be listed item by item in the appendix.

[0126] 3.4 PRM Method Validation PRM Detection and First-Round Quality Screening: For ultra-low input scenarios, due to the extremely limited available materials for a single sample, it is usually difficult to repeatedly collect a large number of independent PRM methods on the same sample. Therefore, it is preferable to first use a reference sample with a higher input volume to perform quality screening on candidate targets. In this embodiment, HeLa cell enzymatic digestion peptides treated with 100 mM sodium lactate for 48 hours were used with a protein input of 200 ng (as an example of a low input sample) and the 19 PRM methods established in Example 3.2 were run on a Stellar mass spectrometer to validate candidate precursors in the ChemIntelligence Library with ultra-low input volume lactylation.

[0127] Data import and screening: Import the data into Skyline for analysis and screen according to the following criteria: absolute peak area > 500; signal-to-background ratio (S / B) > 3; relative abundance > 0.05; time correlation > 0.8; number of effective product ions ≥ 3.

[0128] Parameter settings: Minimum residence time of 10 ms, peak width of 9 seconds, at least 7 acquisition points per peak, acquisition window of 1.4 minutes, and retention of only a single charge state for each precursor, etc., were used. A balanced loading mode was employed to finally compress a compressed lactylated PRM target panel containing 1,224 lactylated peptides (covering 1,533 modification sites and 1,003 proteins), suitable for ultra-low input and single-cell derived samples. Figure 3 a). The 1,224 lactylated peptides together constitute a lactylated PRM target panel suitable for ultra-low input and single-cell derived samples, the specific composition of which is described in Table 3.

[0129] 3.5 Suitability Validation of Single-Cell Derivative Samples To evaluate the applicability of the method of the present invention in samples with extremely low input and samples derived from single cells, it was validated using gradient cell number samples and samples prepared based on a single-cell sorting platform.

[0130] Sample preparation: In one embodiment, HeLa cells treated with 100 mM sodium lactate for 48 hours were digested with 0.25% trypsin-EDTA buffer at 37°C for approximately 4 minutes to prepare a single-cell suspension, and counted using an automated cell counter. The cells were then diluted to 10³, 10⁻⁶, and 10⁻⁶ cells according to a predetermined cell number gradient. 4 and 10 5 Cell samples were collected. Next, the cells were resuspended in 100 μL of lysis buffer, pH 8.5, containing 1% SDC, 10 mM TCEP, 40 mM MCAA, and 100 mM Tris-HCl. The samples were homogenized after heating at 95°C for 5 minutes, centrifuged at 14,000 g for 5 minutes, and the supernatant was collected and enzymatically digested overnight at 37°C. After digestion, formic acid was added to a final concentration of approximately 0.5% to terminate the reaction. The cells were then centrifuged at 16,000 g to remove SDC, desalted using C18 chromatography, and stored at -80°C for later use.

[0131] In another embodiment, for ultra-low input samples of 1–100 cells, the cellenONE single-cell sorting system can be used to sort HeLa cells treated with sodium lactate into 384-well plates. The 384-well plates are pre-filled with 1 μL of master mix containing 0.2% DDM, 100 mM TEAB, and 20 ng / μL trypsin. Sorting can be performed based on cell morphology parameters, for example, selecting cells with a diameter of approximately 15–35 μm and an elongation not exceeding 1.8, and allocating them to the wells according to a predetermined cell number. After sorting, the samples are incubated at 37°C and approximately 70% humidity for approximately 2 hours, then cooled to approximately 20°C, acidified with 3.5 μL of 0.1% TFA, and approximately 4.5 μL of the sample is taken for LC-MS analysis.

[0132] Method application: For 10 4 and 10 5 For cell samples, the protein input for a single test can be controlled to approximately 200 ng using conventional quantitative methods (as an example of a low-input sample); for serially diluted and desalted samples, a conventional LC-MS loading procedure is used; for ultra-low-input samples obtained by cellenONE sorting, loading and detection are performed after acidification.

[0133] In PRM detection, the overall signal intensity gradually decreases as the number of cells decreases. Figure 3 b) The overall CV of the peptide signal increases ( Figure 3 c). Under the single-cell-derived sample preparation and detection conditions of this embodiment, a total of 1,085 lactylated peptides were detected, of which 295 peptides had a CV < 30% ( Figure 3 d). Taking the lactylated peptide AEVEGK(lac)DLPEHAVLK at the K626 site of HNRNPU protein as an example, this peptide was detected in all cell input groups. Figure 3 e), its fragment ion extraction ion chromatogram increases with increasing cell input and remains identifiable in single-cell source samples. Figure 3 f). The above results demonstrate that the panel is feasible for targeted detection of lactylation in single-cell-derived samples under the conditions of this embodiment.

[0134] This embodiment demonstrates that, through the construction of a reference spectral library optimized for ultra-low input scenarios, PRM method scheduling, actual acquisition verification, and quality screening, a compressed PRM target panel for lactylation can be formed, suitable for ultra-low input and single-cell-derived samples. The panel is defined by a clearly defined lactylated modified peptide sequence, the position of the lactylation modification within the peptide, precursor ion and product ion information, and can be annotated with one or more candidate protein accessions and their corresponding lactylation sites and / or protein grouping information in the protein sequences. The panel can be repeatedly invoked as a computer-readable target set and rescheduled to an executable PRM acquisition method based on different cell input levels, liquid chromatography conditions, or mass spectrometry acquisition capabilities. This panel can still achieve lactylated peptide detection in single-cell-derived samples, indicating that it is not merely a one-time detection method parameter, but an executable PRM target panel suitable for targeted detection of ultra-low input samples.

[0135] Table 1. Sub-panel of Lactylated PRM Targets after CV Screening of Lung Cancer-Related Samples Table 2 CD8 + T-cell sample CV screening followed by lactylated PRM target panel Table 3. Lactylated Compressed PRM Target Panel for Ultra-Low Input and Single-Cell Source Samples

Claims

1. A compressed parallel reaction monitoring (PRM) target panel for controlling the acquisition of targeted data on lysine lactylation in a mass spectrometer, characterized in that: The compressed PRM target panel is formed from a large-scale lactylated PRM target panel validated by actual PRM acquisition through one or more rounds of quality screening, and contains multiple lactylated PRM target members. The identity of each member is determined by the modified peptide sequence containing lactylated lysine and the position of the lactylation modification in the modified peptide. The panel also records or can calculate the corresponding precursor ion mass-to-charge ratio and precursor ion charge state for each member. The precursor ion mass-to-charge ratio and precursor ion charge state are acquisition attributes that can be recorded as explicit fields or calculated or converted from the modified peptide sequence, lactylation modification type, and charge state rules. The compressed PRM target panel is stored in a computer-readable form and can be imported, converted, or rescheduled into one or more PRM acquisition methods. When generating the PRM acquisition method, the product ion is selected based on the corresponding lactylated peptide reference spectrum or actual PRM acquisition data to control the mass spectrometer to acquire data from the multiple lactylated PRM targets.

2. A computer-readable PRM target panel for controlling mass spectrometry to perform targeted data acquisition for lysine lactylation, characterized in that, The panel is selected from one or more of the following: (1) Subpanel after CV screening of lung cancer-related samples including all or a subset of the lactylated PRM targets listed in Table 1; (2) CD8, including all or a subset of the lactylated PRM targets listed in Table 2 + Sub-panel after CV screening of T cell samples; (3) Ultra-low input and single-cell source samples of lactylated compressed PRM target panels, including all or a subset of the lactylated PRM targets listed in Table 3. The identity of each panel member is determined by the lactylated modified peptide sequence and the position of the lactylation modification in the peptide. The panel also records or can calculate the precursor ion mass-to-charge ratio and precursor ion charge state for each member, and can be imported, converted or rescheduled to one or more PRM acquisition methods to control the mass spectrometer for data acquisition.

3. A large-scale emulsified PRM target panel, characterized in that, The large-scale lactylated PRM target panel comprises multiple lactylated PRM targets retained after actual PRM data acquisition, peak extraction, and detectability confirmation from the initial lactylated PRM target panel. It can be assigned or rescheduled to one or more PRM acquisition methods to control the mass spectrometer for data acquisition, and can be formed into the compressed lactylated PRM target panel as described in claim 1 or 2 through one or more rounds of quality screening.

4. A lactylated initial PRM target panel, characterized in that, The initial PRM target panel for lactylation is generated based on a reference spectrum library of lactylated peptides or a list of search results for lactylated peptides, and includes multiple candidate lactylated PRM targets that have not yet been retained as members based on actual PRM detection results. The identity of each candidate member is determined by the modified peptide sequence and the position of the lactylation modification within the modified peptide. The panel also records or can calculate the corresponding precursor ion mass-to-charge ratio and precursor ion charge state for each candidate member. The precursor ion mass-to-charge ratio and precursor ion charge state are collected attributes and can be recorded as explicit fields, or calculated or converted from the modified peptide sequence, lactylation modification type, and charge state rules.

5. The emulsified PRM target panel according to any one of claims 1 to 4, characterized in that, The sequence of lactylated modified peptides and the position of lactylation modification in the peptides are used as the basis for judging the identity of panel members; the same panel member can be associated with one or more candidate protein accessions and their corresponding lactylation sites and / or protein grouping information in the protein sequence. The association information is used as a protein mapping annotation and is not a necessary condition for distinguishing panel members. Adjustments to the number or composition of product ions, retention time or retention time window, precursor ion charge state, collision energy, mass-to-charge ratio calibration of precursor or product ions, liquid chromatography conditions, mass spectrometry platform, retention time calibration strategy, PRM acquisition method allocation, software data format, database version, protein merging rules, or peptide-protein mapping relationship do not change the membership of the corresponding target; wherein, when the panel of claim 2 includes a subset of the targets recorded in the corresponding tables in Tables 1 to 3, the subset contains no less than 50, no less than 100, or no less than 500 lactylated PRM targets in the corresponding tables, or contains no less than 30%, no less than 50%, no less than 70%, or no less than 90% of the lactylated PRM targets in the corresponding tables.

6. A method for constructing a compressed emulsified PRM target panel, characterized in that, include: (1) Obtain the lactylated initial PRM target panel as described in claim 4; (2) Dynamically allocate the candidate lactylated PRM targets to one or more PRM acquisition methods based on the chromatographic retention time and the target density in different time intervals; (3) Perform actual PRM data acquisition using the validation sample and perform peak extraction and detectability confirmation to form the large-scale lactylated PRM target panel as described in claim 3; (4) Perform PRM data acquisition on the large-scale lactylated PRM target panel using pre-screened samples; perform one or more rounds of quality screening based on one or more of the following: absolute peak area, signal-to-noise ratio, signal-to-background ratio, relative abundance, time correlation, number of effective product ions, peak shape integrity and coefficient of variation. (5) Retain the lactylated PRM target that meets the predetermined quality requirements to form the compressed lactylated PRM target panel as described in claim 1 or 2; Among the initial panel, large-scale panel, and compressed panel, the member correspondence is maintained by the lactylated modified peptide sequence and the position of the lactylation modification in the peptide. The adjustment of protein mapping annotation and acquisition parameters does not change the member correspondence.

7. A method for controlling a mass spectrometer to perform targeted data acquisition for lysine lactylation, characterized in that, include: (1) Obtaining the lactylated PRM target panel according to any one of claims 1 to 5; (2) Select product ions based on the reference spectrum of the corresponding lactylated peptide or actual PRM data; import, convert or reschedule the panel to one or more PRM acquisition methods; (3) and controlling the mass spectrometer to perform PRM data acquisition according to one or more PRM acquisition methods; the data acquisition is used for lactylation detection, quantitative analysis or cross-sectional comparative analysis of low-input biological samples, clinical tissue samples, immune cell samples, rare cell samples, single-cell level samples or single-cell source samples, and does not include the step of directly determining the disease status or formulating a treatment plan based on the detection results.

8. A non-transitory computer-readable storage medium, characterized in that, The device stores a lactylated PRM target panel as described in any one of claims 1 to 5; the panel can be read, converted, or rescheduled by a processor to generate one or more PRM acquisition methods or to control a mass spectrometer to perform lysine lactylated PRM data acquisition.

9. A system for targeted data acquisition of lysine lactylation, characterized in that, include: Storage module for storing the emulsified PRM target panel as described in any one of claims 1 to 5; A conversion or scheduling module is used to generate one or more PRM acquisition methods based on the panel and the corresponding reference spectrum. And a data acquisition control module, used to control the mass spectrometer to acquire PRM data according to the one or more PRM acquisition methods.

10. The large-scale emulsified PRM target panel according to claim 3, characterized in that, The large-scale lactylated PRM target panel contains no fewer than 15,000 lactylated PRM targets that have been confirmed through actual PRM data acquisition, peak extraction, and detectability testing, and is assigned or rescheduled to no fewer than 10 PRM acquisition methods. Each lactylated PRM target can be traced back to the corresponding candidate target in the initial lactylated PRM target panel based on the lactylated modified peptide sequence and the position of the lactylation modification in the peptide, and has an identifiable chromatographic peak confirmed by peak extraction in actual PRM data. The same panel member can be associated with one or more candidate protein accessions and their corresponding lactylation sites and / or protein grouping information in the protein sequence. The association information is not a necessary condition for distinguishing panel members. The large-scale lactylated PRM target panel is independently stored, retrieved, or rescheduled in a computer-readable form including target member identity information, optional protein mapping annotations, and corresponding peak extraction results.

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