Method for detecting proteins in a sample in a fluidic device using mass spectrometry - Patent Application 20070123333

Abstract

The present disclosure provides methods for detecting and analyzing proteins using mass spectrometry, such as direct matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) for proteins in a fluidic device, or reverse-phase liquid chromatography mass spectrometry (rpLC-MS) for proteins outside of a fluidic device.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 293,502, filed December 23, 2021, and U.S. Provisional Patent Application No. 63 / 432,522, filed December 14, 2022, the disclosures of both of which are incorporated by reference in their entireties herein.

[0002] The present disclosure provides methods for detecting and analyzing proteins using mass spectrometry, such as direct matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) for proteins in a fluidic device, or reverse-phase liquid chromatography mass spectrometry (rpLC-MS) for proteins outside of a fluidic device. [Background technology]

[0003] MALDI-MS and rpLC-MS are widely used techniques to determine the molecular weight (Mw) of biomolecules and have a very good capacity to rapidly determine the Mw of small molecules, peptides, and proteins. Mass spectrometry imaging, such as MALDI-MS imaging, is used on thin tissue sections fixed on a MALDI-MS target plate. Many analytical techniques are based on the force exerted by a light beam (known as photomanipulation), enabling interactive biology at the cellular level and thus opening up new opportunities for drug discovery. Photomanipulation, which enables highly selective and dynamic processes in micro- and nanoscale systems, has proven to be a versatile and integrative technique across many scientific disciplines. The technique is based on light-induced electrical motion that produces specified forces in both solid and fluidic structures (1). For example, commercially available fluidic devices such as the integrated technology of the Berkeley Lights (BLI) Beacon® Optofluidic System (Emeryville, CA) have the flexibility and power to address a wide range of applications applicable to commercial large molecule drug development, including antibody discovery, clonal selection, gene editing, phenotype-genotype matching, and cell line development. RPLC-MS is also a widely used technique to determine the Mw of pharmaceutical and biopharmaceutical related molecules (12). Summary of the Invention [Means for solving the problem]

[0004] There is a need to develop "on-microchip" assays that predict the performance of molecules in shake flasks and bioreactors. Disclosed herein are methods for using mass spectrometry, such as MALDI-MS, on proteins in fluidic devices. Additionally, the disclosed methods allow protein-producing cells to be grown in the fluidic device and mass spectrometry to be performed directly on the medium containing the protein, such as conditioned medium.

[0005] In one aspect, disclosed herein is a method of detecting proteins in a fluidic device. In various embodiments, the method includes mass spectrometry of the sample while the sample is in the fluidic device. In some embodiments, the proteins in the sample are reduced. The methods disclosed herein provide methods of detecting protein levels and identifying changes in the molecular weight (Mw) of the protein or analyzing modifications to the protein. For example, if the protein is an antibody, the method allows for the determination of the Mw of the heavy and light chains of the antibody, etc., from antibody-producing cells grown on-chip, along with identification of clipping, light and heavy chain pairing and mispairing (e.g., in the case of multispecific mAbs), and other post-translational modifications.

[0006] In some embodiments, the disclosure provides a method for detecting proteins in a sample in a fluidic device comprising: contacting a sample in the device with solid supports each comprising a ligand for a protein, whereby the ligand binds to the protein of the sample, and each solid support comprising a ligand bound to the protein comprises a unique barcode that is distinct from other solid supports comprising a ligand bound to the protein; transferring the solid supports comprising the ligand bound to the protein from a first location in the fluidic device to a second location, where the second location is within the fluidic device or is external to the fluidic device; and mass spectrometry of the sample at the second location.

[0007] In various embodiments, the method for detecting proteins further comprises reducing the proteins in the sample before subjecting the sample to mass spectrometry.For example, the proteins in the sample can be reduced by contacting the proteins with sinapic acid and TCEP, for example by mixing the proteins in the sample with TCEP in the presence of sinapic acid.For example, the proteins can be reduced in situ or on a mass spectrometry plate, such as a MALDI plate, before subjecting the sample to mass spectrometry.

[0008] The methods of the present disclosure can be carried out using any type of mass spectrometry capable of electrospray ionization (ESI). For example, mass spectrometry for use in the methods described herein includes matrix-assisted laser desorption / ionization time-of-flight / time-of-flight (MALDI-TOF / TOF), liquid chromatography mass spectrometry / mass spectrometry (rpLC-MS), hydrophobic interaction chromatography mass spectrometry (HIC-MS), or cation exchange chromatography mass spectrometry (CEX-MS). In related embodiments, the rpLC-MS includes electrospray ionization (ESI).

[0009] In any of the disclosed methods, the sample is any fluid or preparation that contains a protein. In various embodiments, the sample is a fluid that contains the protein to be processed, measured, or analyzed for stability and / or structural integrity or other attributes. In some embodiments, the sample comprises or consists of conditioned medium, or any fluid from which a protein is purified or isolated. In some embodiments, the sample comprises cells that produce the protein.

[0010] In any of the disclosed methods, the protein comprises or consists of an antibody, an antibody protein product, a bispecific T cell engager (BiTE®) molecule, an antibody fragment, an antibody fusion peptide or antigen-binding fragment thereof, a peptide, a growth factor, or a cytokine. In related embodiments, the antibody is a polyclonal antibody or a monoclonal antibody. As used herein, the term "antibody protein product" refers, in various instances, to any one of several antibody surrogates that are based on the structure of an antibody but are not found in nature. In some aspects, the antibody protein product has a molecular weight in the range of at least about 12 kDa to 1 MDa, e.g., at least about 12 kDa to 750 KDa, at least about 12 kDa to 250 kDa, or at least about 12 kDa to 150 kDa. In certain embodiments, antibody protein products have a valency (n) ranging from monomers (n=1) to dimers (n=2), trimers (n=3), tetramers (n=4), if not higher valencies. Antibody protein products, in some embodiments, are based on the complete antibody structure and / or mimic antibody fragments that retain complete antigen-binding ability, such as scFv, Fab, and VHH / VH (discussed below). The smallest antigen-binding antibody fragment that retains an intact antigen-binding site is the Fv fragment, consisting exclusively of the variable (V) region. Soluble, flexible amino acid peptide linkers are used to link the V region to the scFv (single chain fragment variable) fragment to stabilize the molecule, or constant (C) domains are added to the V region to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab fragments can be readily produced in host cells, such as prokaryotic host cells. Other antibody protein products include disulfide bond stabilized scFv (ds-scFv), single chain Fab (scFab), and dimeric and multimeric antibody types such as diabodies, triabodies, and tetrabodies, which include different types containing scFv linked to oligomerization domains, or minibodies (miniAbs). The smallest fragments are the VHH / VH of camelid heavy chain Abs and single domain Abs (sdAbs).The building block most frequently used to create new antibody formats is the single chain variable (V) domain antibody fragment (scFv), which contains V domains (VH and VL domains) from heavy and light chains linked by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of peptibodies contains a bioactive peptide grafted onto the Fc domain. Peptibodies are well described in the art. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012). Other antibody protein products include single chain antibodies (SCAs); diabodies; triabodies; tetrabodies; bispecific or trispecific antibodies, etc. Bispecific antibodies can be classified into five major classes: BsIgG, adducted IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015). In an exemplary embodiment, the antibody protein product comprises or consists of a Bispecific T Cell Engager (BiTE®) molecule, which is an artificial bispecific monoclonal antibody. A BiTE® molecule is a fusion protein that contains two scFvs of different antibodies; one binds to CD3 and the other binds to the target antigen. BiTE® molecules are known in the art. See, e.g., Huehls et al., Immuno Cell Biol 93(3):290-296 (2015); Rossi et al., MAbs 6(2):381-91 (2014); Ross et al., PLoS One 12(8):e0183390.

[0011] In various embodiments, the protein is partially purified before being subjected to mass spectrometry. The term "purification" refers to the isolation or separation of a protein from components of a mixture that contains the protein, including crude materials, cell lysates, conditioned media, or other cell culture materials that contain the protein. The term "partial purification" refers to the removal of some, but not all, of the components of a mixture that contains the protein. Components that are removed from the mixture include cell debris, protein aggregates, lipids, and / or proteases.

[0012] In some embodiments, the method of the present disclosure further comprises partially purifying the protein. The purification step can be performed using any method known in the art. For example, partial purification can include contacting the sample with a solid support (e.g., beads) comprising a ligand for the protein prior to subjecting the sample to mass spectrometry. As used herein, a "ligand" for a protein refers to a chemical entity that binds to the protein, such as an antibody or binding partner that binds to the protein. For example, if the protein comprises or consists of a cytokine, examples of ligands for purposes herein include the corresponding cytokine receptor (or binding fragments thereof). For example, if the protein comprises or consists of an antibody, examples of ligands for purposes herein include an antigen for that antibody, an anti-idiotypic antibody, an anti-Fc antibody, protein A, or protein G. In exemplary embodiments, the solid support (e.g., beads) comprises an anti-FC protein, protein A, or protein G, or the solid support (e.g., beads) comprises protein A or protein G.

[0013] In some embodiments, the solid support comprising a ligand bound to the protein comprises a unique barcode, and the partial purification further comprises other solid supports comprising ligands bound to other proteins and comprising other barcodes different from the unique barcode.

[0014] In some embodiments, mass spectrometry of samples in the methods of the disclosure includes performing on a batch that includes solid supports, where the solid support that includes a ligand bound to a protein includes a unique barcode, and the solid support further includes at least some other solid supports that include ligands bound to other proteins and that include other barcodes. The term "batch" refers to multiple solid supports that include samples from multiple wells or containers.

[0015] For example, partial purification may include contacting the sample with a solid support (e.g., beads) comprising a ligand for the protein prior to mass spectrometry of the sample. In some embodiments, partial purification further includes transferring the solid support (e.g., beads) comprising the ligand bound to the protein from a first location in the fluidic device to a second location in the fluidic device prior to mass spectrometry of the sample. The second location may be a second fluidic chip, a second fluidic plate, or a second region, flow path, channel, chamber, or pen in the fluidic device. Additionally, the second location is outside the fluidic device, for example, a well in a multiwell plate such as a 96-well plate or a 384-well plate.

[0016] In any of the methods of the present disclosure, the method can be performed using any fluidic device or apparatus known in the art. A fluidic device (or fluidic apparatus) is a device that includes one or more independent circuits configured to hold fluid, each circuit being composed of fluidically interconnected circuit elements. Circuit elements include, but are not limited to, regions, flow paths, channels, chambers, and / or pens, and at least one port configured to allow fluid to enter or exit the fluidic device. A fluidic circuit can be configured to have a first end fluidly connected to a first port (e.g., an inlet) of a microfluidic device, and a second end fluidly connected to a second port (e.g., an outlet) of the fluidic device, or to a second fluidic device, or to a second region, flow path, channel, chamber, or pen of a fluidic device. The fluidic device can be a microfluidic device, although other scales, such as nanoscale, are also suitable. For example, the fluidic device can be a microfluidic chip, a microfluidic channel, a microfluidic cell, a nanofluidic chip, a nanofluidic channel, a nanofluidic cell, or an isolation pen. The separate circuit or circuits of the fluidic device may include silicon, such as a silicon surface configured to allow a sample to be placed on it. For example, the separate circuit or circuits may include a flow region defined by at least one silicon surface. By way of example, the separate circuit or circuits may include a microfluidic or nanofluidic channel defined by at least one silicon surface. In some embodiments, the microfluidic or nanofluidic channel is etched into the silicon. It will be appreciated that mass spectrometry may be performed on a silicon surface (see, for example, Lewis et al., "Desorption / ionization on silicon (DIOS) mass spectrometry: background and applications." International Journal of Mass Spectrometry 226 (2003) 107-116). Thus, in some embodiment methods, a sample is placed on a silicon surface of the fluidic device.

[0017] In a microfluidic device, the circuit includes a flow region, which may include a microfluidic channel, and at least one chamber, and holds a volume of fluid of less than about 1 mL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 μL. In certain embodiments, the microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 μL. The microfluidic circuit may be configured to have a first end fluidly connected to a first port (e.g., an inlet) in the microfluidic device and a second end fluidly connected to a second port (e.g., an outlet) in the microfluidic device.

[0018] As used herein, a "nanofluidic device" or "nanofluidic apparatus" is a type of fluidic device having a fluid circuit including at least one circuit element configured to hold a volume of fluid less than about 1 μL, for example, about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less. A nanofluidic device may include a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). In certain embodiments, one or more (e.g., all) of the at least one circuit element are configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In another embodiment, one or more (e.g., all) of the at least one circuit element are configured to hold a volume of fluid of about 20nL-200nL, 100-200nL, 100-300nL, 100-400nL, 100-500nL, 200-300nL, 200-400nL, 200-500nL, 200-600nL, 200-700nL, 250-400nL, 250-500nL, 250-600nL, or 250-750nL.

[0019] As used herein, a "fluid channel" or "flow channel" refers to a flow region of a fluidic device that has a length that is significantly longer than both the horizontal and vertical dimensions. For example, a flow channel may be at least 5 times longer, e.g., at least 10 times longer, at least 25 times longer, at least 100 times longer, at least 200 times longer, at least 500 times longer, at least 1,000 times longer, at least 5,000 times longer, or more, than either the horizontal or vertical dimension. In some embodiments, the length of the flow channel ranges from about 50,000 microns to about 500,000 microns, including any range therebetween. In some embodiments, the horizontal dimension ranges from about 100 microns to about 1,000 microns (e.g., about 150 microns to about 500 microns) and the vertical dimension ranges from about 25 microns to about 200 microns, e.g., about 40 microns to about 150 microns. It is noted that flow channels may have a variety of different spatial configurations within a fluidic device and are therefore not limited to being completely linear elements. For example, a flow channel can include one or more sections that have any of the following configurations: curved, bent, spiral, slanted, descending, branching (e.g., multiple different flow paths), and any combination thereof. In addition, a flow channel can have different cross-sectional areas along its path, widening or narrowing to allow the flow of a fluid of interest therethrough.

[0020] The present disclosure also provides a MALDI mass spectrometry target plate comprising a fluidic device attached to a surface of the MALDI mass spectrometry target plate. In some embodiments, the fluidic device is open, whereby the interior of the fluidic device is configured for direct engagement with a mass spectrometer. For example, the mass spectrometry used in the methods described herein includes matrix-assisted laser desorption / ionization time-of-flight / time-of-flight (MALDI-TOF / TOF) or liquid chromatography mass spectrometry / mass spectrometry (LC-MS / MS). In related embodiments, the LC-MS / MS includes electrospray ionization.

[0021] Further, the present disclosure provides a system for detecting proteins in a sample, comprising any of the mass spectrometry plates disclosed herein. In some embodiments, the system further comprises a control or protein standard. In addition, in various embodiments, the system further comprises a solid support (e.g., a bead) comprising a ligand such as anti-Fc protein, protein A, or protein G.

[0022] The present disclosure also provides a kit comprising the mass spectrometry plate disclosed herein. In various embodiments, the kit further comprises a solid support (e.g., beads) comprising a ligand, such as an anti-Fc protein, Protein A, or Protein G. [Brief description of the drawings]

[0023] [Figure 1] Mass spectra from MALDI-MS analysis of proteins spotted on a MALDI plate. The top row shows the mass spectrum of 1 mg / mL protein, the middle row shows the mass spectrum of 0.5 mg / mL protein, and the bottom row shows the mass spectrum of 0.125 mg / mL protein. [Diagram 2] Mass spectrum from MALDI-MS analysis of conditioned medium containing expressed mAb. The spectrum is dominated by the light chain, while the signal corresponding to intact antibody (indicated by the arrow) is suppressed. [Figure 3A] RP-HPLC chromatogram from isolation of day 11 conditioned medium containing expressed antibody showing that three major peaks are detected in the conditioned medium containing expressed antibody. [Figure 3B] Reducing SDS-PAGE identifying the eluted RP-HPLC fractions, showing reduced SDS-PAGE identification of RP-HPLC fractions from peaks 3a and 3b, which are size-matched to the purified mAb control in the last lane. [Figure 4A]1 shows the rpLC-MS ESI spectrum from analysis of the RP-HPLC eluate of peak 1 by LC-ESI-MS, identifying peak 1 as a mixture of cysteinylated and glutathionylated light chains. [Figure 4B] 1 shows the rpLC-MS ESI spectrum from analysis of the RP-HPLC eluate of peak 2 by LC-ESI-MS, identifying peak 2 as a light chain dimer and a dimer with cysteine ​​or glutathione adducts. [Diagram 5] RP-HPLC chromatograms and ESI spectra from conditioned media analysis after affinity purification using a Protein A / G spin column are shown. Top: Recovery of expressed mAb from conditioned media using a Protein A / G spin column showing the light chain derivative eluting in the unbound fraction. Bottom: RP-HPLC confirming the presence of mAb in the bound fraction after elution with 1% acetic acid. [Figure 6] RP-HPLC chromatograms showing 80% recovery of mAb from conditioned media by binding to Pierce Protein A / G Magnetic Beads. The top panel shows the abundance of mAb in conditioned media before addition of magnetic beads (peak 3). The bottom panel shows the decrease in abundance of mAb in conditioned media after addition of magnetic beads (decrease in peak 3), demonstrating that most of the mAb was bound to the beads. [Figure 7] MALDI-MS spectrum from direct analysis of Protein A / G magnetic beads with bound mAb, showing the Mw of intact mAb. [Figure 8] MALDI-MS spectrum from on-plate reduction of mAb bound to Pierce Protein A / G magnetic beads. [Figure 9]RP-HPLC chromatograms showing recovery of mAb from conditioned media by binding to Promega Protein A beads. Peak 1 is the mAb light chain. Peak 2 is the mAb light chain dimer. Peak 3 is the mAb of interest. The top panel shows the abundance of mAb in the conditioned media before addition of Protein A beads. The bottom panel shows the decrease in the abundance of mAb in the conditioned media after addition of Protein A beads (dramatic decrease in peak 3), demonstrating that most of the mAb was bound to the beads. [Figure 10] MALDI-MS spectrum from direct analysis of Promega Protein A magnetic beads with bound mAb, showing the Mw of the intact mAb molecules. [Figure 11] MALDI-MS spectrum of mAb after acid elution from Protein A magnetic beads prior to analysis. [Figure 12] Overlaid spectra obtained from direct analysis of mAb bound to beads, showing a 3-fold increase in signal by including an acid elution step prior to analysis. [Figure 13] MALDI-MS of mAb after reduction directly on the glass cover of the MALDI-MS plate is shown. [Figure 14] 1 shows the surface of the BLI chip attached to a MALDI-MS target plate. [Figure 15] A, shows the BLI chip mounted in the back pocket ("picture frame") of a MALDI-MS target plate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Mass spectrometry imaging, such as MALDI-MS imaging, is commonly used on thin tissue sections fixed on a MALDI-MS target plate. Disclosed herein is a method for detecting proteins in a sample by mass spectrometry of the sample while it is within a fluidic device. In the present methods, systems, and apparatus, the MALDI-MS target plate can be modified to accommodate / mount a BLI chip. For example, MALDI lasers are typically 10-20 μm in diameter (special focus), and microfluidic pens are approximately 60 μm wide, so the dimensions of the laser "fit" within the BLI pen.

[0025] If one could analyze / measure the Mw of molecules such as mAbs and / or multispecifics by MALDI-MS directly from the BLI pen, one could determine if the molecule is the expected molecule based on the measured Mw. One could potentially identify the levels of chain pairing and mispairing, as well as post-translational modifications (PTMs). The accuracy and sensitivity of the mass spectrometry could be improved by reducing the mAb on the target plate, or the antibody could be reduced in situ (e.g., on a solid substrate), or the antibody could be reduced directly on the mass spectrometry plate.

[0026] In some embodiments, the mass spectrometry plate is configured to accommodate a fluidic device. For example, a fluidic device, a fluidic chip, or a fluidic chamber or channel, or an isolation pen may be mounted or machined on the mass spectrometry plate. For example, the mass spectrometry plate may be configured to mount a fluidic device thereon. In some embodiments, the fluidic device is mounted on the surface of the MS plate or MS matrix. In another embodiment, the fluidic device is mounted in a back pocket of the mass spectrometry plate. In these embodiments, the fluidic device may be open or may include a removable cover for inserting the MS matrix and / or allowing access by the mass spectrometer. The removable cover may include a glass, crystal, or polymeric cover slip.

[0027] In another embodiment, the liquid MS matrix is ​​poured directly into the fluidic device. For example, if the MS is a MALDI-MS and the fluidic device is a tip or an isolation pen, the liquid MALDI matrix is ​​poured directly into the tip or pen using a microfluidic system such as the BLI Beacon® microfluidic system. In the case of a fluidic chip that includes a removable cover, the cover can be removed before or after the MS matrix is ​​poured into it.

[0028] The fluidic device allows for the growth and proliferation of single cells in a chamber or isolation pen, which allows for clonal selection of cells producing the protein of interest. Clonal selection allows for the selection of clones for large-scale protein production and purification during drug discovery and biopharmaceutical manufacturing, e.g., antibody production. Mass spectrometry allows for the analysis of protein modifications, such as post-translational modifications that change the Mw of the protein of interest, e.g., glycosylation, cysteinylation, glutathionylation, oxidation, deamination, glycation, phosphorylation, sulfation, or ubiquitination. Furthermore, mass spectrometry allows for the determination of mispaired species and fragmented proteins. The disclosed method also allows for the continuous analysis of cells while they are growing, and assays can be repeated on the same growing cells.

[0029] A colony of biological cells is a "clone" if all viable cells capable of reproduction in the colony are daughter cells derived from a single progenitor cell. In certain embodiments, all daughter cells in a clonal colony are derived from a single parent cell by 10 or fewer divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single progenitor cell by 14 or fewer divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single progenitor cell by 17 or fewer divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single progenitor cell by 20 or fewer divisions. The term "clonal cells" refers to cells of the same clonal colony.

[0030] As used herein, a "colony" of biological cells refers to two or more cells (e.g., about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or more than 1000 cells).

[0031] As used herein, the term "maintaining cells" refers to providing an environment, including both fluid and gaseous components, and optionally a surface, that provides the conditions necessary for cells to remain viable and / or grow.

[0032] As used herein, the term "proliferation" when referring to cells refers to an increase in cell number.

[0033] mass spectrometry In an exemplary embodiment, the method includes detecting proteins in a sample using mass spectrometry. Disclosed herein is a method for mass spectrometry of a sample while the sample is in a fluidic device. Mass spectrometry is used to measure the mass / charge of ions. The method herein can include ESI-enabled mass spectrometry.

[0034] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) is a simple, effective and widely used technique for determining the Mw of biomolecules, with excellent capabilities for rapid Mw determination of small molecules (2), peptides, and proteins (3). It was first reported in 1985 by Karas and Hillenkamp, ​​in which MALDI was used to analyze a selection of amino acids and dipeptides (4). In various embodiments, the method comprises MALDI-MS.

[0035] MALDI-MS imaging has the ability to analyze many different tissue specimens / samples. Briefly, thin tissue sections are fixed to a MALDI-MS target plate, coated with a matrix, and ablated with a laser (in a mass spectrometer). For detection, time-of-flight (ToF) analyzers are typically used, but MALDI has been coupled with both ion cyclotron resonance (ICR) (5) and Orbitrap (6) MS systems. A large variety of analytes, ranging from proteins, peptides, protein modifications, small molecules, drugs and their metabolites, pharmaceutical ingredients, endogenous cellular metabolites, lipids, and other analytes, are made accessible by MALDI-MS imaging of thin tissue sections (7). This method is label-free and allows for the simultaneous multiplex analysis of hundreds to thousands of molecules in the same tissue section. In various embodiments, the mass spectrometry comprises or consists of MALDI-MS. In various embodiments, the mass spectrometry comprises or consists of matrix-assisted laser desorption / ionization time-of-flight / time-of-flight (MALDI-TOF / TOF). MALDI TOF / TOF is an efficient method for detecting fragmented peptides. In various embodiments, the mass spectrometry comprises or consists of liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), also called liquid chromatography-tandem mass spectrometry. In this disclosure, the two terms MALDI-TOF / TOF and MALDI-LIFT-TOF / TOF are used interchangeably.

[0036] sample The sample may contain any type of protein that can be measured, processed, or analyzed for stability and / or structural integrity. In some embodiments, the sample comprises or consists of conditioned media or any liquid from which proteins can be purified or isolated. In various embodiments, the protein sample so subjected to the methods disclosed herein comprises or consists of large peptides, antibodies, antibody fragments, antibody fusion peptides, or antigen-binding fragments thereof. In related embodiments, the antibody is a polyclonal or monoclonal antibody.

[0037] In various embodiments, the proteins in the sample are reduced. The proteins are reduced to improve sensitivity. The proteins are reduced using any method known in the art. For example, the proteins are reduced with redox agents such as dithiothreitol (DTT), β-mercaptoethanol, and TCEP (tris(2-carboxyethyl)phosphine) in sinapinic acid. Additionally, the proteins are reduced by mixing the proteins in the sample with TCEP and TCEP in sinapinic acid or by contacting the proteins in the sample with acetic acid. For example, the proteins are released from the solid support (e.g., beads) with acetic acid, and then TCEP and sinapinic acid are added. The proteins can be reduced before being inserted into the mass spectrometry plate, or the proteins can be reduced after being inserted into the mass spectrometry plate.

[0038] Fluid Devices A fluidic device refers to an apparatus that uses small amounts of fluid to perform various types of analysis. Fluidic devices include one or more independent circuits configured to hold fluid, with each circuit being made up of fluidically interconnected circuit elements. Circuit elements include, but are not limited to, regions, flow paths, channels, chambers, and / or pens, and at least one port configured to allow fluid to enter or exit the fluidic device. These devices use chips, cells, channels, or isolated pens that contain the fluid for analysis.

[0039] Microfluidic devices typically have one or more channels with at least one dimension less than 1 mm. Common fluids used in microfluidic devices include whole blood samples, bacterial cell suspensions, protein or antibody solutions, and various buffers. Microfluidic devices can be used to obtain a variety of measurements, such as molecular diffusion coefficients, fluid viscosity, pH, chemical binding coefficients, and enzyme reaction rates. Other applications of microfluidic devices include capillary electrophoresis, isoelectric focusing, immunoassays, flow cytometry, sample injection of proteins for analysis by mass spectrometry, PCR amplification, DNA analysis, cell manipulation, cell separation, cell patterning, and chemical gradient formation. Many of these applications are useful in clinical diagnostics.

[0040] Advantages of using microfluidic devices include that the volume of fluid in these channels is very small, typically a few nanoliters, and the amount of reagents and test substances used is rather small. Furthermore, when analyzing protein-producing cells, a relatively small number of cells (even a single cell) can produce sufficient amounts and concentrations of protein for analysis, and the culture time for colony growth can be reduced or avoided. The manufacturing techniques used to build microfluidic devices, which will be described in more detail later, are relatively inexpensive and very suitable for mass production, both for very sophisticated multiplexed devices. Microfluidic technology allows the production of highly integrated devices to perform several different functions on the same substrate chip.

[0041] Any fluidic device, including commercially available devices, can be used (or modified to be used) in the methods of the present disclosure. The fluidic device can be configured for use in an optofluidic system that can use light to manipulate materials in the fluidic device, such as cells. An exemplary microfluidic device is described herein as a chip that includes a Berkley Lights (BLI) pen. For example, the BLI pen can be analyzed with the Beacon® Optofluidic System, the Lightning™ Optofluidic System, or the Culture Station System (BLI. Emeryville, CA). Another exemplary optofluidic system is the Cyto-Mine® system (Sphere Fluidics, Great Abington, Cambridge, UK).

[0042] In any of the methods of the present disclosure, a single cell or a small number of cells (approximately 1-10) are seeded into the fluidic device and these cells express the protein to be detected. The proteins expressed by the cells in the fluidic device are directly sampled by MS while in the fluidic device using any of the methods of the present disclosure. In this scenario, the proteins expressed in the fluidic device need to be immobilized and / or coated with a MALDI matrix prior to MS analysis. The fluidic device, such as a BLI chip, is then loaded onto an existing MALDI-MS target plate and then loaded into the MS instrument for subsequent analysis. Depending on how the overexpressed proteins in the fluidic device are handled (presence or absence of reducing agent in the matrix), the proteins can be analyzed under either reducing or non-reducing conditions. For example, if the protein to be analyzed is a monoclonal antibody, the monoclonal antibody can be analyzed under either reducing (light and heavy chains) or non-reducing (intact) conditions.

[0043] In various embodiments, the MS performed in the disclosed method is MALDI-MS. Due to the special focus of the MALDI laser, this type of MS is well suited to efficiently perform the disclosed method. Another feature to consider in the disclosed method is the dimensions of the fluidic device, e.g., the isolation pen. Modern lasers in MALDI MS instruments are typically solid-state nitrogen-based Nd-YAG / YLF (neodymium-doped yttrium aluminum garnet / yttrium lithium fluoride) lasers, which have been documented to achieve laser spot diameters between 1.1 μm and 8.4 μm (8, 9). Available microfluidic chip dimensions include 130×370 μm, 50×370 μm, 40×200 μm, and 40×160 μm (these sizes are commercially available from BLI, Emeryville, CA). Thus, based on the dimensions of available isolation pens, modern MALDI-MS lasers can easily "fit" into current pens and be used for ablation / analysis of matrix-anchored proteins. Furthermore, current repetition rates of modern lasers are up to 5 kHz (10), so that most commonly available fluidic chips (40 × 160 μm) can be fully analyzed in a few hours.

[0044] Partial protein purification and sample pooling In some embodiments, the disclosed method includes partially purifying the proteins in the sample before subjecting the sample to MS. The partial purification can be performed by any method known in the art. For example, the partial purification can include contacting the sample with a solid support, such as beads or resin, that includes a ligand for the protein before subjecting the sample to mass spectrometry. The ligand for the protein can be a chemical that binds to the protein, such as an antibody or binding partner that binds to the protein. In an exemplary embodiment, the protein is a monoclonal antibody, the solid support is a bead that includes an anti-Fc protein, protein A, or protein G, and the partial purification includes transferring the bead that includes the ligand bound to the antibody from a first location in the fluidic device to a second location in the fluidic device before subjecting the sample to MS.

[0045] The solid supports (e.g., beads) are removed from the fluidic device and transferred to another MS target plate or 96-well and / or 384-well plates for (reverse-phase liquid chromatography-mass spectrometry) rpLC-MS analysis (11, 12). Transferring the proteins captured on the solid supports (e.g., beads) from the fluidic device allows for "pooling" and thus improves MS sensitivity. As the mobile phase of rpLC-MS is acidic, it is compatible with eluting the captured proteins from the solid supports (e.g., beads).

[0046] Additionally, in any of the methods of the present disclosure, MS may be tandem MS (both MALDI-MS / MS and rpLC-MS / MS) performed to obtain or confirm protein sequence information and / or to identify or quantitate post-translational modifications. "Top-down MS / MS" refers to MS / MS performed on non-reduced samples. "Middle-down MS / MS" refers to MS / MS performed on reduced material. "Bottom-up MS / MS" refers to MS / MS performed on reduced and then proteolytically digested material.

[0047] Suitable solid supports for purifying or partially purifying proteins include, for example, beads or resins. When the solid support is a bead, the bead can be of a size such that a single bead can contain an amount of protein for MS. For example, as shown in the examples herein, a bead having an average diameter of at least about 1 μm can contain a sufficient amount of protein for analysis by MS. For example, the solid support can comprise or consist of beads having an average diameter of at least about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 10 μm, 20 μm, or 40 μm, including ranges between any two of the values ​​listed, such as 1-5 μm, 1-10 μm, 1-20 μm, 1-40 μm, 5-10 μm, 5-20 μm, 5-40 μm, 10-20 μm, or 10-40 μm. For any solid support, a ligand for the protein can be immobilized on the solid support, for example, by covalent attachment. In some methods, the beads can be magnetic beads. When the solid support is a resin, the resin can be a polymeric resin such as cellulose, polystyrene, agarose, and polyacrylamide or agarose.

[0048] In some methods, MS can be performed on a batch of solid supports, each containing a different protein. For example, single beads containing a ligand can be coupled to a protein at each separate location of a fluidic device, such as a pen, such that each single bead contains a protein produced by a different clone or colony of clone cells. The single beads can then be pooled at a second location and analyzed at that second location (either in the fluidic device or external to the fluidic device). In such methods, the beads associated with each separate location (e.g., a pen) can be barcoded such that the protein derived from the clone at that location can be identified from the pool. For example, the protein at each location can contain a unique peptide or PTM barcode, or each bead or ligand thereon can contain a unique barcode. Examples of suitable barcodes include peptides or nucleic acids with unique sequences or molecular weights, dyes or combinations of dyes, glycans and / or carbohydrates or combinations thereof, or fluorophores or combinations of fluorophores. In some embodiments, the beads are sized such that each separate location on the fluidic device (e.g., a pen) containing proteins of different clones can accommodate only a single bead. For example, each bead can have a diameter at least half the diameter of the independent location such that no more than one bead is placed at an independent location at a time. Each bead can include a unique barcode. In some embodiments, beads are barcoded with a location that includes a protein. For example, each location (e.g., a pen) can include a unique peptide or nucleic acid barcode (produced by a cell or cloned cell at that location), which is immobilized on a bead in the system.

[0049] Some methods include a sample in a device having solid supports, each solid support including a ligand for a protein. The ligand can bind to a protein of the sample, and each solid support including a ligand bound to a protein can include a unique barcode that is different from other solid supports including a ligand bound to a protein. The solid supports including the ligand bound to the protein can be moved from a first location to a second location in the fluidic device. MS (either MALDI-MS or LC-MS) can be performed on the protein at the second location. The second location can be within the fluidic device, for example, an open area of ​​the fluidic device, or an area including a removable cover, so that the proteins in that area can be analyzed by MS (either MALDI-MS or LC-MS). Alternatively, the second location can be outside the fluidic device, for example, a well in a multi-well plate such as a 96-well plate or a 384-well plate. The solid supports can be analyzed in batches by MS at the second location. The solid supports can be used to determine the clonal origin of the protein of each bead. EXAMPLES

[0050] Basic method Protein A / G magnetic beads (catalog 88802) were purchased from Thermo Fisher. Magne® Protein A beads (catalog G8781) were obtained from Promega.

[0051] Partial purification of mAbs using magnetic beads. The magnetic bead (20 μL) slurry was transferred to a 1.5 mL microcentrifuge tube and subsequently washed with 500 μL of PBS. After mixing using a vortex mixer, the tube was placed on a magnetic stand and the beads were allowed to settle as a "thin band". The liquid was removed and 100 μL of conditioned medium was added to the beads followed by gentle shaking at room temperature for 30-60 min. After the beads had settled, the liquid portion (unbound sample) was removed and analyzed using RP-HPLC. The beads were then washed with 500 μL of PBS. After the beads had settled, the liquid portion (washing solution) was carefully removed. 20 μL of PBS was added to the remaining beads containing the bound mAb and mixed to generate a slurry. The slurry (1 μL) was spotted onto a MALDI plate followed by the addition of 2 μL of matrix (10 mg / mL sinapinic acid in 0.1% TFA 50% acetonitrile) and 1 μL of 5% acetic acid. The spots were allowed to dry at room temperature prior to MALDI-MS.

[0052] On-plate reduction of mAb The beads containing the bound mAb were placed on a MALDI plate and the beads were contacted with sinapinic acid (2 μL) (as above) and 20 mM TCEP. The samples and their reduction products were allowed to dry at room temperature. MALDI-MS was then performed on the dried spots.

[0053] Alternatively, mAbs were diluted in 1% acetic acid (10 μL) and 0.1 M TCEP. Samples were incubated at 37° C. for 20 min, then placed on a MALDI plate (1 μL) and allowed to dry at room temperature. Sinapic acid (1 μL) was overlaid onto the spot, air-dried, and mass determined by MALDI-MS.

[0054] MALDI-MS equipment MALDI-MS was performed using a Bruker UltrafleXtreme TOF / TOF MALDI mass spectrometer. The instrument was equipped with a 2000 Hz Smartbeam II (Nd:YAG) laser operated in linear positive mode and calibrated using BSA as an external standard. The accelerating voltage used was set to 25 kV. The laser power was optimized and typically 5000-8000 laser shots were collected per sample.

[0055] All LC-MS data were acquired on an Agilent 6224 TOF LC / MS system with a 1290 Infinity LC system. Chromatographic separation was achieved using a Zorbax SB300-C8 3.5 μm 2.1 × 50 mm column operated at a temperature of 70 °C. The solvents used were as follows: mobile phase A was water containing 0.1% v / v TFA. Mobile phase B was 90% n-propanol containing 0.1% v / v TFA. Initial gradient conditions were 0.0-1.0 min, 20% mobile phase B; 1.0-9.0 min, 20-70% mobile phase B; 9.0-10.0 min, 70-100% mobile phase B, held at 100% for an additional 1 min. The flow rate was 0.4 mL / min.

[0056] MALDI-MS target plate engineering to incorporate BLI chips Two options were developed for mounting the BLI chip to the MALDI plate. First, the BLI chip was mounted on the surface of a pocket milled into the front of the MALDI plate. The depth of the pocket was such that the surface of the BLI chip matched the front of the MALDI chip. The BLI chip was secured in the pocket by two M2 screws that passed through existing holes in the BLI chip and screwed into tapped holes in the MALDI plate (Figure 12).

[0057] The BLI chip is then attached to a pocket at the rear of the MALDI plate. The perimeter of the pocket is not milled completely through the MALDI plate so that the BLI chip is captured in a "picture frame". The center of the pocket is milled through the MALDI plate so that the front of the BLI chip is exposed and the surface of the microfluidic component matches the surface of the MALDI plate (Figure 14). The BLI chip was secured in the pocket when the MALDI plate was attached to the MALDI plate carrier.

[0058] Example 1: Evaluation of MALDI-MS for clone assessment An experiment was set up to determine whether MALDI-MS in combination with a fluidic device could be used for clone characterization. First, the mAb was diluted to 1 mg / mL in 1x PBS, then 5 μL of protein was mixed with 5 μL of 1x PBS to serially dilute to 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL. MALDI was performed as described above. For MALDI plate spotting, 1 μL of each protein concentration was mixed with 1 μL of sinapinic acid (10 mg / mL in 50% MeCN in 0.1% TFA), after which 1 μL was spotted onto a MALDI plate, air-dried, and the Mw was measured. This corresponds to spotting 0.5 μg, 0.25 μg, 0.125 μg, and 0.0625 μg of mAb onto a well. The data is shown in the mass spectrum in Figure 1. No "good spots" were obtained with the 0.25 mg / mL material, and the lower row is a broader peak. This experiment demonstrates that mAbs can be observed on MALDI-MS plates and that this method can be used in combination with a fluidic device to detect mAbs.

[0059] Example 2 Evaluation of MALDI-MS for direct monitoring of mAb expression in conditioned medium Conditioned medium from cells expressing a monoclonal antibody, designated here as mAb2, was analyzed using MALDI-MS as described above. Conditioned medium was harvested after 11 days of culture, spotted directly onto a MALDI plate, matrix was added, the sample was air-dried, and the Mw was measured.

[0060] As shown in Figure 1, the mass spectrum of the non-reduced sample shows that the mass spectrum is dominated by the light chain signal, while the intact mAb signal is suppressed (indicated by the arrow). To determine what is interfering with the intact mAb signal, the conditioned medium at day 11 was analyzed using RP-HPLC and LC-ESI-MS. RP-HPLC analysis of the conditioned medium showed its complexity, including free cysteinylated light chains, free glutathionylated light chains, light chain dimers, and material identified as Ab. Figure 2A shows the RP-HPLC chromatogram, in which the conditioned medium resolved into three major peaks (peaks 1, 2, 3a, and 3b). Figure 2B shows the reduced SDS-PAGE analysis of the RP-HPLC fractions showing that the expressed mAb eluted in peak 3.

[0061] Figures 3A and B show LC-ESI-MS data identifying RP-HPLC fractions from peaks 1 and 2. Figure 3A shows that peak 1 contains a mixture of cysteinylated and glutathionylated light chains. Figure 3B shows that peak 2 corresponds to light chain dimers and dimers containing either cysteine ​​or glutathionylated adducts. Thus, analysis of the conditioned medium showed a complex mixture enriched in overexpressed light chains (free cysteinylated light chains, free glutathionylated light chains, light chain dimers), mAb, and polymer-like material. Co-elution of polymer-like material with mAb in peak 3 of the RP-HPLC fractions is likely the reason for the failure of the Mw measurement of mAb. Therefore, a preliminary purification step is beneficial to analyze mAb expression in conditioned medium by MALDI-MS.

[0062] Example 3 MALDI-MS analysis of mAbs after partial purification steps To improve the sensitivity of the MALDI-MS analysis, the protein was partially purified before subjecting the samples to MALDI-MS. Day 11 conditioned medium was contacted with a Protein A / G spin column (Pierce) as described previously. Figure 4 shows that the expressed mAb was recovered from the conditioned medium using a Protein A / G spin column. The top chromatogram shows that the light chain derivative was recovered in the unbound fraction, while the bound mAb was detected in the 1% acetic acid eluate, whose Mw is shown in the bottom ESI spectrum. Thus, Protein A / G magnetic beads were used for a pre-purification step of the mAb suitable for subsequent characterization by MALDI-MS.

[0063] The beads were added to the conditioned medium to capture the mAb, which was then collected using a magnetic stand, washed, mixed with 20 μL of PBS, and 1 μL of the slurry was spotted onto a MALDI plate. This was followed by the addition of 2 μL of sinapinic acid MALDI matrix and 1 μL of 5% acetic acid to elute the bound mAb, which was then analyzed by RP-HPLC and LC-ESI-MS. The spots were then air-dried and subjected to MALDI-MS. Figure 5 shows the RP-HPLC chromatograms showing that Protein A / G magnetic beads recovered up to 80% of the mAb from the conditioned medium. The lower red chromatogram is the RP-HPLC analysis. Figure 6 shows the mass spectrum of the mAb bound to Protein A / G magnetic beads and analyzed directly by MALDI-MS.

[0064] To test whether the mAb could be detected after reduction directly on the beads, 20 mM TCEP was added to the matrix added to the beads spotted on the MALDI plate. Reduction was performed at room temperature and the spots were air-dried before MALDI-MS. Figure 7 shows that the light chain was detected and the signal from the heavy chain was suppressed. This is probably due to glycosylation, which reduced the ion signal and the peak from the intact mAb due to incomplete reduction of the mAb (which can be further optimized). In Figure 7, the 49300 ion is the 3+ ion of the mAb. The 98636 peak is (Fab)2. The 23436 ion is the light chain. These relative amounts do not indicate relative percentages. These data show that the components of the mAb bound to the beads can be easily analyzed directly after reduction on-plate.

[0065] The bead experiment described above was repeated using Promega Protein A beads under the same experimental conditions. Again, the reduction in peak 3 indicates that most of the mAb in the conditioned medium was bound to the beads (Figure 8). Direct analysis of the beads showed similar results to those using Protein A / G beads described above (see Figure 9). In a separate experiment, 2 μL of beads with bound mAb were transferred to a microcentrifuge tube and 5 μL of 5% acetic acid was added. After the beads had settled, 1 μL of the solution was subjected to MALDI-MS, which gave a signal three times more intense (see Figures 10 and 11). Thus, partial purification of mAb from conditioned medium using Protein A magnetic beads can be readily employed prior to analysis by MALDI-MS.

[0066] Example 4 MALDI-MS analysis of mAbs after reduction on the glass cover of the MALDI-MS plate An assay was performed to determine whether mAbs could be analyzed by MALDI-MS after direct reduction of the mAbs on the glass cover of a MALDI-MS plate. The mAbs were diluted in 10 μL of 50% acetonitrile containing 1% acetic acid and 0.1 M TCEP, and 1 μL of the slurry was spotted onto the MALDI plate. Sinapic acid (10 mg per mL of 50% acetonitrile containing 0.1% TFA) was then layered directly onto the mAB spot on the glass cover and allowed to air dry before running MALDI-MS.

[0067] Figure 13 shows that the light chain was detected and the signal from the heavy chain was suppressed, possibly due to glycosylation, which reduced the ion signal and the peak from the intact mAb due to incomplete reduction of the mAb, which can be further optimized. The 22798.1 peak is the light chain and the 50495.7 peak is the heavy chain. These relative amounts do not indicate the relative percentages. These data demonstrate that the components of the mAb can be easily analyzed directly after on-plate reduction.

[0068] Example 5 MALDI-MS screening of antibodies / plasmocytes in a microfluidic device. The proof-of-concept data from the previous examples showing that MALDI-MS can be used to accurately analyze the Mw of mAbs directly in conditioned medium and on Protein A / G beads under reducing and non-reducing conditions indicates that the MALDI-MS assay can be adapted to score secreted antibodies on BLI chips mounted on MALDI target plates.

[0069] Mounting the BLI chip onto the MALDI target plate: The spatial focus of the MALDI laser is 10-20 µm in diameter, while the width of the BLI pen is approximately 60 µm, so the dimensions of the laser "fit" inside the BLI pen. Antibody-producing cells are introduced into the microfluidic device by flowing the suspension into the inlet and stopping the flow when the cells are located within the flow region / microfluidic channel. The cells are then loaded into the isolation pen, targeting one cell per pen. The cells are moved from the flow region / microfluidic channel to the separation region of the isolation pen using photoactivated DEP forces (OEP technique).

[0070] Assaying cells using MALDI-MS: A few days after pen loading, cells are assayed to analyze the molecular weight of the secreted antibody, which is then compared to the expected molecular weight to determine if the correct antibody is expressed. Additionally, MALDI-MS analysis allows for the detection of chain mispairing and the identification of post-translational modifications. Reduction of the antibody on the plate / chip improves mass accuracy and sensitivity.

[0071] The Fc and / or ProA / G captured beads are removed from the BLI pen and transferred to another MALDI-MS target plate or to a 96-well and / or 384-well plate for reversed-phase liquid chromatography mass spectrometry (11, 12) rpLC-MS analysis. Transferring the bead-captured mAbs from the BLI tip / pen allows for "pooling" and therefore improved MS sensitivity. Optionally, beads can be barcoded to allow identification of the clone of origin of the protein on each bead. The mobile phase of the rpLC-MS is acidic and therefore compatible with eluting the captured mAbs from the anti-Fc and / or ProA / G beads.

[0072] References 1. Jorgolli,M.;Nevill,T.;Winters,A.;Chen,I.;Chong,S.;Lin,F.F.;Mock,M.;Chen,C.;Le,K.;Tan,C.;Jess,P.;Xu,H.;Hamburger,A.;Stevens,J.;Munro,T.;Wu,M.;Tagari,P.;Miranda,L.P.,Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring.Biotechnol Bioeng 2019,116 (9),2393-2411. 2. Cohen,L.H.;Gusev,A.I.,Small molecule analysis by MALDI mass spectrometry.Anal Bioanal Chem 2002,373 (7),571-86. 3. Cohen,S.L.;Chait,B.T.,Influence of matrix solution conditions on the MALDI-MS analysis of peptides and proteins.Anal Chem 1996,68 (1),31-7. 4. Karas,M.;Bachmann,D.;Hillenkamp,F.,Influence of the Wavelength in High-Irradiance Ultraviolet Laser Desorption Mass Spectrometry of Organic Molecules.Analytcal Chemistry 1985,57,2935-2839. 5. Buck,A.;Ly,A.;Balluff,B.;Sun,N.;Gorzolka,K.;Feuchtinger,A.;Janssen,K.P.;Kuppen,P.J.;van de Velde,C.J.;Weirich,G.;Erlmeier,F.;Langer,R.;Aubele,M.;Zitzelsberger,H.;Aichler,M.;Walch,A.,High-resolution MALDI-FT-ICR MS imaging for the analysis of metabolites from formalin-fixed,paraffin-embedded clinical tissue samples.J Pathol 2015,237(1),123-32. 6. Korte,A.R.;Yandeau-Nelson,M.D.;Nikolau,B.J.;Lee,Y.J.,Subcellular-level resolution MALDI-MS imaging of maize leaf metabolites by MALDI-linear ion trap-Orbitrap mass spectrometer.Anal Bioanal Chem 2015,407(8),2301-9. 7.Aichler,M.;Walch,A.,MALDI Imaging mass spectrometry:current frontiers and perspectives in pathology research and practice.Lab Invest 2015,95(4),422-31. 8. Guenther,S.;Koestler,M.;Schulz,O.;Spengler,B.,Laser spot size and laser power dependence of ion formation in high resolution MALDI imaging.Internation Journal of Mass Spectrometry 2010,294(1),7-15. 9. Zavalin,A.;Yang,J.;Hayden,K.;Vestal,M.;Caprioli,R.M.,Tissue protein imaging at 1 mum laser spot diameter for high spatial resolution and high imaging speed using transmission geometry MALDI TOF MS.Anal Bioanal Chem 2015,407(8),2337-42. 10. Prentice,B.M.;Chumbley,C.W.;Caprioli,R.M.,High-speed MALDI MS / MS imaging mass spectrometry using continuous raster sampling.J Mass Spectrom 2015,50(4),703-10. 11. Dillon,T.M.;Bondarenko,P.V.;Rehder,D.S.;Pipes,G.D.;Kleemann,G.R.;Ricci,M.S.,Optimization of a reversed-phase high-performance liquid chromatography / mass spectrometry method for characterizing recombinant antibody heterogeneity and stability. J Chromatogr A 2006,1120(1-2),112-20. 12. Campuzano,I.D.G.;Sandoval,W.,Denaturing and Native Mass Spectrometric Analytics for Biotherapeutic Drug Discovery Research: Historical,Current,and Future Personal Perspectives.J Am Soc Mass Spectrom 2021,32(8),1861-1885.

Claims

1. 1. A method for detecting proteins in a sample comprising mass spectrometry of the sample while the sample is in a fluidic device.

2. 1. A method for detecting a protein in a sample in a fluidic device, comprising: contacting a sample in the device with solid supports each comprising a ligand for said protein, whereby said ligand binds to said protein in the sample, and each solid support comprising said ligand bound to said protein comprises a unique barcode distinct from other solid supports comprising said ligand bound to said protein; transferring the solid support containing the ligand bound to the protein from a first location within the fluidic device to a second location, the second location being within the fluidic device or being external to the fluidic device; and mass analyzing the sample at the second location; The method includes:

3. The method of claim 1 or 2, wherein the protein in the sample is reduced.

4. 3. The method of claim 1 or 2, further comprising reducing the proteins in the sample prior to subjecting the sample to mass spectrometry.

5. The method of claim 3 or 4, wherein the protein is reduced in situ or the protein is reduced in the mass spectrometer.

6. The method according to any one of claims 1 to 5, wherein the mass spectrometry is matrix-assisted laser desorption mass spectrometry / ionization time-of-flight / time-of-flight (MALDI-TOF / TOF).

7. 7. The method of any one of claims 1 to 6, wherein the mass spectrometry is liquid chromatography mass spectrometry / mass spectrometry (rpLC-MS / MS), hydrophobic interaction chromatography mass spectrometry (HIC-MS), or cation exchange chromatography mass spectrometry (CEX-MS).

8. The method of claim 7, wherein the LC-MS / MS comprises electrospray ionization.

9. The method of any one of claims 1 to 8, wherein the sample comprises conditioned medium.

10. The method according to any one of claims 1 to 9, wherein said protein comprises or consists of a large peptide, an antibody, an antibody fragment, an antibody fusion peptide or an antigen-binding fragment thereof.

11. The method of claim 10, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

12. The method of any one of claims 1 to 11, wherein the protein is partially purified before being subjected to mass spectrometry.

13. The method of any one of claims 1 to 11, further comprising partially purifying the protein.

14. 14. The method of claim 13, wherein said partially purifying comprises contacting said sample with a solid support comprising a ligand for said protein prior to subjecting said sample to mass spectrometry.

15. 15. The method of claim 14, wherein the solid support comprising the ligand bound to the protein comprises a unique barcode, and wherein the partially purifying further comprises other solid supports comprising ligands bound to other proteins and comprising other barcodes different from the unique barcode.

16. 16. The method of claim 15, wherein mass analyzing the sample comprises performing on a batch comprising the solid support, the solid support comprising the ligand bound to the protein comprises a unique barcode, and the solid support further comprises at least some other solid supports comprising ligands bound to other proteins and comprising other barcodes.

17. 17. The method of any one of claims 14 to 16, wherein the partially purifying further comprises transferring the solid support comprising the ligand bound to the protein from a first location within the fluidic device to a second location within the fluidic device prior to mass spectrometry of the sample.

18. 17. The method of any one of claims 14 to 16, wherein the partially purifying further comprises transferring the solid support comprising the ligand bound to the protein from a first location in the fluidic device to a second location on a multiwell plate prior to mass spectrometry of the sample.

19. The method of any one of claims 14 to 18, wherein the solid support comprises an anti-FC protein, protein A, or protein G, or the solid support comprises protein A or protein G.

20. The method of claim 2, wherein the solid support comprises an anti-FC protein, protein A, or protein G, or the solid support comprises protein A or protein G.

21. The method according to any one of claims 1 to 20, wherein the fluidic device is a microfluidic chip or an isolation pen.

22. The method of any one of claims 1 to 21, wherein the fluidic device comprises a silicon surface and the sample is disposed on the silicon surface.

23. A mass spectrometry plate comprising a fluidic device attached to a surface of the mass spectrometry plate.

24. 24. The mass spectrometry plate of claim 23, wherein the fluidic device is open, whereby an interior of the fluidic device is configured for direct engagement with a mass spectrometer.

25. The mass spectrometry plate according to claim 23 or 24, wherein the mass spectrometry is matrix-assisted laser desorption / ionization time-of-flight / time-of-flight MALDI-TOF / TOF).

26. The mass spectrometry plate of claim 23 or 24, wherein the mass spectrometry is liquid chromatography mass spectrometry / mass spectrometry (rpLC-MS / MS), hydrophobic interaction chromatography mass spectrometry (HIC-MS), or cation exchange chromatography mass spectrometry (CEX-MS).

27. 27. The mass spectrometry plate of claim 26, wherein the LC-MS / MS comprises electrospray ionization.

28. A system for detecting proteins in a sample, comprising a mass spectrometry plate according to any one of claims 23 to 27.

29. 30. The system of claim 28, further comprising a control or protein standard.

30. A kit comprising the mass spectrometry plate according to any one of claims 23 to 29.

31. The system or kit of any one of claims 28 to 30, further comprising a solid support comprising a ligand such as anti-FC protein, protein A, or protein G.

32. 32. The method of any one of claims 2, 14-20, or the system or kit of claim 31, wherein the solid support comprises beads, such as beads having an average diameter of at least about 1 μm.

Images