This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.

The authors have nothing to disclose.

Absolute protein quantification is essential for a very diverse range of biomedical applications such as biomarker validation and signal transduction pathway modeling. Recently, targeted proteomics using LC-SRM has benefited from improvements to numerous technologies including peptide standard preparation, HPLC, QqQ-MS, and LC-SRM data analysis. Consequently, it has become a powerful alternative to immunoassays. Immunoassays can be extremely sensitive and high-throughput, but developing a robust immunoassay can be extrem...

Proteomic experiments that use mass spectrometry (MS) can be designed to use either non-targeted (shotgun) or targeted methods. Discovery proteomics generally relies on bottom-up shotgun MS, either by using a traditional data-dependent acquisition mode, or by using one of the recently developed data-independent techniques (e.g., MSE, SWATH)1,2. Shotgun proteomics is a powerful tool for high-throughput peptide identification and relative quantification, but it is generally unsuitable for absolute quantification or for targeting small, defined sets (~tens) of proteins. The MS method most often used for targeted proteomics is selected reaction monitoring (SRM) because of its high sensitivity, speed, and dynamic range3-5. Alternatives to SRM include parallel reaction monitoring, which takes advantage of high-resolution, full MS scanning6.
SRM is usually performed using a nano-flow reversed-phase high-performance liquid chromatography (nano-RP-LC) instrument coupled to a nano-electrospray ionization (nano-ESI) ion source attached to a triple quadrupole mass spectrometer (QqQ-MS). In a typical experiment, sample proteins are proteolytically digested, and the resulting peptides are chromatographically separated, desorbed, and ionized. The resulting precursor ions are m/z filtered by the first quadrupole (Q1) and fragmented in the second quadrupole (q2) by colliding them with a collision gas. The resulting fragment ions are m/z-filtered in the third quadrupole (Q3) and quantified by a dynode. Each precursor and fragment ion pair is referred to as a transition, and each transition is monitored for a specified time period (the dwell time; typically 2-50 msec). During LC-SRM, the QqQ-MS cycles through a predefined list of transitions (the duty cycle is typically &#8804;3 sec), and a chromatogram of each transition is produced.
Alternative strategies for protein quantification typically use immunoassays such as dot blots, Western blots, ELISAs, antibody microarrays, reverse phase protein microarrays, microfluidic immunoassays, digital ELISAs, and microsphere-based immunoassays7. The best immunoassays can be significantly more sensitive than LC-SRM, and sample throughput of immunoassays can be significantly higher than that of LC-SRM5. However, developing immunoassays can be expensive and/or time consuming, and the resulting assays can be vulnerable to cross-reactivity and/or interference, incompatible with cell/tissue lysis/homogenization methods, and/or not amenable to multiplexing5,8. Some of these issues can be addressed by coupling antibody- and MS-based techniques. For example, target proteins can be enriched using immunoprecipitation prior to proteolysis and LC-SRM9-12. Alternatively, the SISCAPA technique employs immunoprecipitation subsequent to proteolysis at the peptide level13,14. In addition to immunoenrichment strategies, immunodepletion of high abundance proteins can be employed to increase LC-SRM sensitivity by reducing interference by coeluting analytes15,16.
MS-based protein quantification can be divided into relative and absolute quantification, and also into label-free and stable isotope labeling (e.g., metabolic labeling, chemical labeling, and heavy-labeled protein and peptide internal standards). Label-free techniques can be useful for relative protein quantification, but are unsuitable for accurate absolute quantification. By comparison, labeling techniques have reduced error associated with sample preparation and MS variance, and are often used for relative protein quantification17. For example, stable isotope labeled proteome (SILAP) standards prepared using a cultured human cell line enabled relative quantification of potential biomarkers via LC-SRM of human serum18. Accurate absolute protein quantification by MS requires that purified, quantified, isotopically-labeled protein or peptide internal standards be spiked-into biological samples prior to MS. The incorporation of heavy isotope labeled internal standards into an LC-SRM workflow enables absolute quantification that has been shown to be highly reproducible and transferable between laboratories16,19.
Stable isotope labeled internal standards for absolute protein quantification by MS include peptide standards prepared using solid phase synthesis20, proteins composed of concatenated protease-cleavable peptide standards21, and full-length protein standards22. Target protein covalent modification and incomplete sample preparation (i.e., incomplete sample lysis and homogenization, and incomplete protein solubilization, denaturation, alkylation, and proteolysis) can undermine accurate quantification. Internal protein standards are the least likely to be affected by most of these potential problems, but they are usually the most difficult to prepare. An alternative is to analyze each target protein using multiple internal peptide standards which are designed to include amino- and carboxy-terminal native flanking residues. Regardless of which type of internal standard is employed, it should be spiked-into the biological samples at as early a point during sample preparation as possible. Also, multiple sample preparation techniques (e.g., different denaturation conditions) should be tested. The usage of multiple orthogonal experimental techniques (experimental cross-validation) is a viable strategy for overcoming most potential quantification challenges23-25.
LC-SRM quantification of proteins is a highly flexible technique that has been used in a wide variety of applications. Notably, it has been used to study peptide and protein biomarkers within clinical samples such as serum, core biopsies, and fine needle aspirates5. LC-SRM has also been used to measure the stoichiometry of protein complexes5,26, to detect botulinum neurotoxins27, to quantify protein phosphorylation dynamics within signaling pathways5, and to quantify changes in protein conformation28.
Our laboratory is using LC-SRM to quantify the signaling proteins that mediate macrophage chemotaxis to support the development of chemotaxis pathway simulations. The overall scheme of the protocol (Figure 1) begins with ranking the tentative target peptides. Subsequently, crude external peptide standards are synthesized and used to develop LC-SRM assays for qualitative analyses of biological samples. If the biological sample-derived target peptide is detected, purified heavy-labeled internal peptide standards are prepared for quantitative LC-SRM. This protocol can be used to accurately quantify proteins from a wide variety of biological samples, and to support investigations of a wide variety of protein targets.

<table><tbody><tr><td>Acetonitrile (ACN), LC-MS grade</td><td>Fisher</td><td>A955-1</td><td></td></tr><tr><td>BCA (bicinchoninic acid) protein assay kit</td><td>Fisher</td><td>23235</td><td></td></tr><tr><td>Beads for bead beating, zirconia-silica, 0.1 mm</td><td>BioSpec Products</td><td>11079101z</td><td></td></tr><tr><td>Bestatin hydrochloride</td><td>Sigma</td><td>B8385-10MG</td><td></td></tr><tr><td>Cell culture DMEM (with glucose, without L-glutamine)</td><td>Lonza</td><td>12-614F</td><td></td></tr><tr><td>Cell culture EDTA, 500 mM, pH8</td><td>Gibco</td><td>15575</td><td></td></tr><tr><td>Cell culture fetal bovine serum (FBS)</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>Cell culture L-glutamine</td><td>Sigma</td><td>G8540-25G</td><td></td></tr><tr><td>Cell culture phosphate buffered saline (PBS) pH 7.4</td><td>Gibco</td><td>10010-049</td><td></td></tr><tr><td>Cell culture Trypan Blue viability stain, 0.4% w/v</td><td>Lonza</td><td>17-942E</td><td></td></tr><tr><td>Cellometer Auto T4 cell counter</td><td>Nexcelom Bioscience</td><td>Cellometer Auto T4</td><td></td></tr><tr><td>Cellometer Auto T4 disposable counting chambers</td><td>Nexcelom Bioscience</td><td>CHT4-SD100-014</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma</td><td>D5545-5G</td><td></td></tr><tr><td>Formic acid, LC-MS grade, ampules</td><td>Fisher</td><td>A117-10X1AMP</td><td></td></tr><tr><td>Hemocytometer, Neubauer-improved, 0.1 mm deep</td><td>Marienfeld-Superior</td><td>0640030</td><td></td></tr><tr><td>HEPES, 1 M, pH 7.2</td><td>Mediatech</td><td>25-060-CI</td><td></td></tr><tr><td>Hydrochloric acid, 37% w/w</td><td>VWR</td><td>BDH3028-2.5LG</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma</td><td>I1149-5G</td><td></td></tr><tr><td>Laser Based Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-2000</td><td></td></tr><tr><td>LC coated silica capillary, 50 &amp;micro;m id</td><td>Polymicro Technologies</td><td>1068150017</td><td></td></tr><tr><td>LC vial, autosampler, 12 mm x 32 mm polypropylene</td><td>SUN SRI</td><td>200-268</td><td></td></tr><tr><td>LC vial screw cap, autosampler, pre-slit PTFE/silicone</td><td>SUN SRI</td><td>500-061</td><td></td></tr><tr><td>Luciferase, from Photinus pyralis</td><td>Sigma</td><td>L9506-1MG</td><td></td></tr><tr><td>Pepstatin A</td><td>EMD Millipore</td><td>516481-25MG</td><td></td></tr><tr><td>pH strips colorpHast (pH 0.0-6.0)</td><td>EMD Chemicals</td><td>9586-1</td><td></td></tr><tr><td>PhosStop phosphatase inhibitor cocktail</td><td>Roche</td><td>04906837001</td><td></td></tr><tr><td>RapiGest SF</td><td>Waters</td><td>186001861</td><td></td></tr><tr><td>Sep-Pak SPE, C18 1 ml 100 mg cartridge</td><td>Waters</td><td>WAT023590</td><td></td></tr><tr><td>Sep-Pak SPE, extraction manifold, 20 position</td><td>Waters</td><td>WAT200609</td><td></td></tr><tr><td>Sep-Pak SPE, flat-surfaced rubber bulb</td><td>Fisher</td><td>03-448-25</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher</td><td>S318-500</td><td></td></tr><tr><td>SpeedVac vacuum concentrator</td><td>Fisher</td><td>SPD111V</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA), LC-MS grade</td><td>Fisher</td><td>A116-50</td><td></td></tr><tr><td>Trypsin, sequencing grade, modified</td><td>Promega</td><td>V5113</td><td></td></tr><tr><td>Tube decapper for Micronic tubes</td><td>USA Scientific</td><td>1765-4000</td><td></td></tr><tr><td>Tubes, 2 ml microcentrifuge, o-ring screw-cap, sterile</td><td>Sarstedt</td><td>72.694.006</td><td></td></tr><tr><td>Urea</td><td>Sigma</td><td>U0631-500g</td><td></td></tr><tr><td>Water, LC-MS grade</td><td>Fisher</td><td>W6-1</td><td></td></tr></tbody></table>

selected reaction monitoring mass spectrometry for absolute protein quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

The development of predictive computational models of signal transduction pathways is one of the fundamental goals of systems biology53. Unfortunately, even for signaling pathways that have been studied extensively and have a high clinical significance, it is still not generally possible to quantitatively predict pathway behavior in response to perturbations (e.g., this is true for the MAPK/ERK pathway54). Recently, an investigation employed targeted proteomics, transcriptomics, and computa...

Watch this Scientific Journal Video about Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification at JoVE.com

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

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16.9K Views.  National Institutes of Health. This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Video: Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Biochemistry

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Chemistry

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

A protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM is a convenient method for multiplexed targeted proteomics analysis of small numbers of cells, including single cells. It capitalizes on using excessive exogenous protein as a carrier and high-specificity LC-SRM for targeted quantification.

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent ...

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.