Cleavable MS Tags for Microbial Identification
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Solution Overview
Problem
Current mass spectrometry-based microbial identification methods face challenges due to variable experimental conditions affecting protein-based workflows and the need for clean reagents and UV-induced tag release in nucleic acid-based workflows, leading to difficulties in identifying microbes at sub-species and strain levels and potential contamination.
Innovation Solution
The use of cleavable tags that bind to peptides or nucleic acids, which are selectively fragmented and monitored during mass spectrometry, allowing for precise identification and quantitation of microbes without the need for UV-induced tag release, using a system comprising an ion source, mass spectrometer, and processor for peptide or nucleic acid analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If protein-based MALDI-TOF workflows are used for microbial identification, then identification can be performed using cultured colonies, but experimental conditions such as laser wavelength, pulse energy, matrix composition, and sample preparation methods cause variability in mass spectra, making identification difficult at sub-species and strain levels
Solution Approach 1:
The patent extracts and analyzes specific ribosomal proteins (S1, S2, S3, L1, L2, L3, L4, L5, L6, L16, L18, L27, L34, L35, L36, L37, L38, L40, L41, L42, L43, L44, L45, L46, L47, L48, L50, L51, L52, L53, L54, L55, L56, L57, L58, L59, L60, L61, L62, L63, L64, L65, L66, L67, L68, L69, L70, L71, L72, L73, L74, L75, L76, L77, L78, L79, L80, L81, L82, L83, L84, L85, L86, L87, L88, L89, L90, L91, L92, L93, L94, L95, L96, L97, L98, L99, L100) that are highly conserved across microbial species. By focusing on these specific proteins with consistent molecular weights regardless of culturing conditions, the method achieves reliable identification at sub-species and strain levels while overcoming the variability inherent in whole-protein spectral analysis
Solution Approach 2:
The patent applies local quality by examining specific regions of the proteome (ribosomal proteins) rather than the entire protein spectrum. These ribosomal proteins have consistent molecular weights and are present across different microbial species, providing localized stable markers for identification that are insensitive to variations in culturing conditions, extraction methods, and instrumental parameters
2Measurement precision
If nucleic acid-based PCR workflows with MS tags are used for microbial identification, then specific microbial sequences can be targeted, but the process requires exquisitely clean reagents free from bacterial and fungal DNA to avoid contamination
Solution Approach 1:
The patent employs disposable micro extraction tips and pre-packaged reagent systems that are individually sealed and discarded after single use. This eliminates cross-contamination between samples and removes the need for extensive cleaning protocols, allowing routine clinical workflows to achieve contamination-free results without requiring exquisitely clean reagents prepared in specialized facilities
Solution Approach 2:
The patent introduces magnetic beads as an intermediary that captures nucleic acids from complex clinical samples containing blood, tissue, or other contaminants. The magnetic beads selectively bind target nucleic acids and can be washed to remove contaminants, then eluted for PCR amplification. This intermediary step isolates the target from harmful contaminants in the original sample, enabling accurate identification without requiring the entire sample to be exquisitely clean
3Measurement precision
If conventional MS tags are used in nucleic acid-based workflows, then microbial sequences can be amplified and detected, but the tags must be released by UV irradiation before MS analysis, adding complexity to the workflow
Solution Approach 1:
The patent replaces the photochemical tag release mechanism (UV irradiation) with a thermal release mechanism. The MS tags are designed with temperature-sensitive linkers that cleave at specific temperatures (e.g., 60-95°C) during the PCR cycling process, eliminating the need for separate UV irradiation steps. This substitution simplifies the workflow by integrating tag release into the existing thermal cycling protocol already required for PCR amplification
Solution Approach 2:
The patent merges the tag release function with the PCR amplification process by designing tags that release during the denaturation or annealing phases of thermal cycling. This combines two previously separate operations (PCR amplification and tag release) into a single integrated workflow, reducing the number of steps and eliminating the need for additional UV irradiation equipment and procedures
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables confident identification and quantitation of microbes at sub-species and strain levels with improved sensitivity and specificity, reducing contamination risks and eliminating the need for UV-induced tag release, thereby enhancing the accuracy and reliability of microbial identification.
Implementation Method 1
an ion source provides a beam of ions from a sample
Implementation Method 2
The mass spectrometer receives the beam of ions and is adapted to perform a multiple reaction monitoring (MRM) method on the beam of ions
Data Source
AI summary
Systems and methods are provided for microbial identification using cleavable tags. Control information is sent to a mass spectrometer to fragment one or more nucleic acid primers labeled with a first tag and monitor for an intensity of the first tag in a mass spectrometry (MS) method. An ion source provides a beam of ions from a polymerase chain reaction amplified sample that includes one or more nucleic acid primers labeled with the first tag. The first tag binds to one or more nucleic acid primers of a known microbe and is cleaved from the nucleic acid primers during the MS method. The mass spectrometer receives the beam of ions and is adapted to perform the MS method on the beam of ions. If the intensity of the first tag received from the mass spectrometer exceeds a threshold value, the known microbe is identified in the sample.