Mass spectrometry quantifies target engagement for a KRASG12C inhibitor in FFPE tumor tissue.

BACKGROUND: Quantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development. However, frozen tissues are often not available from clinical trials so it is critical that assays are applicable to formalin-fixed, paraffin-embedded (FFPE) tissues in order to extend mass spectrometry-based target engagement studies into clinical settings. METHODS: Wild-type RAS and RASG12C was quantified in FFPE tissues by a highly optimized targeted mass spectrometry assay that couples high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) with internal standards. In a subset of samples, technical reproducibility was evaluated by analyzing consecutive tissue sections from the same tumor block and biological variation was accessed among adjacent tumor regions in the same tissue section. RESULTS: Wild-type RAS protein was measured in 32 clinical non-small cell lung cancer tumors (622-2525 amol/µg) as measured by FAIMS-PRM mass spectrometry. Tumors with a known KRASG12C mutation (n = 17) expressed a wide range of RASG12C mutant protein (127-2012 amol/µg). The variation in wild-type RAS and RASG12C measurements ranged 0-18% CV across consecutive tissue sections and 5-20% CV among adjacent tissue regions. Quantitative target engagement was then demonstrated in FFPE tissues from 2 xenograft models (MIA PaCa-2 and NCI-H2122) treated with a RASG12C inhibitor (AZD4625). CONCLUSIONS: This work illustrates the potential to expand mass spectrometry-based proteomics in preclinical and clinical oncology drug development through analysis of FFPE tumor biopsies.

Global Extraction from Parallel Reaction Monitoring to Quantify Background Peptides for Improved Normalization and Quality Control in Targeted Proteomics.

Mass spectrometry-based targeted proteomics employs heavy isotope-labeled proteins or peptides as standards to improve accuracy and precision. The input sample amount is often determined by the total quantity of endogenous proteins or peptides, as defined by spectrophotometric assays, before the heavy-isotope standards are spiked into the samples. Errors in spectrophotometric measurements, which may be due to low sensitivity or chemical or biological interference, have a direct impact on the quantitative mass spectrometry results. Currently used targeted proteomics workflows cannot identify or correct deviations that arise from differences in the input sample amount. We have developed a workflow, global extraction from parallel reaction monitoring (PRM), to identify and quantify thousands of background peptides that are inherently acquired by PRM experiments. These background peptides were used to identify differences in the input sample amount and to reduce this variance by intensity-based, post-acquisition normalization. This approach was then applied to a xenograft study to improve the quantification of human proteins in the presence of mouse tissue contamination. In addition, these background peptides also provided a direct source of quality control metrics related to sample handling and preparation.

Targeted data-independent acquisition for mass spectrometric detection of RAS mutations in formalin-fixed, paraffin-embedded tumor biopsies.

Genomic testing for KRAS and NRAS mutations in clinical biopsies of various cancers is routinely performed to predict futility of anti-epidermal growth factor receptor (anti-EGFR) therapies. We hypothesized that RAS mutations could be detected and quantified at the protein level for diagnostic purposes using data-independent acquisition (DIA)-based mass spectrometry in formalin-fixed, paraffin-embedded (FFPE) tumor samples. We developed a targeted DIA assay that surveys the specific mass range of all possible peptides harboring activating mutations in KRAS exon 2. When the assay was applied to tumor samples with known KRAS or NRAS mutations (G12A, G12D, G12V, and G13D), RAS-mutant and wild-type peptides were successfully detected in 11 of 13 biopsy samples. Mutation statuses obtained by DIA were concordant with those obtained by DNA sequencing, and yields of mutant peptide (mutant peptide/[mutant + wild-type peptides]) exhibited linear correlation with yields of RAS-mutant mRNA. When applied to biopsy samples with failed DNA testing results, the DIA assay identified an additional RAS-mutated sample. SIGNIFICANCE: Proteomic detection of RAS mutations by DIA in tumor biopsies can provide solid evidence of mutant RAS protein regardless of the mutation types and sites in exon 2. This robust method could rescue samples that fail genomic testing due to insufficient tumor tissue or lack of sequenceable DNA. It can be used to explore the relationship between protein expression level of mutant RAS and therapeutic outcome.

Immuno-Matrix-Assisted Laser Desorption/Ionization Assays for Quantifying AKT1 and AKT2 in Breast and Colorectal Cancer Cell Lines and Tumors.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway is one of the most commonly dysregulated signaling pathways that is linked to cancer development and progression, and its quantitative protein analysis holds the promise to facilitate patient stratification for targeted therapies. Whereas immunohistochemistry (IHC) and immunoassays are routinely used for clinical analysis of signaling pathways, mass spectrometry-based approaches such as liquid chromatography/electrospray ionization multiple reaction monitoring mass spectrometry (LC/ESI-MRM-MS) are more commonly used in clinical research. Both technologies have certain disadvantages, namely, the nonspecificity of IHC and immunoassays, and potentially long analysis times per sample of LC/ESI-MRM-MS. To create a robust, fast, and sensitive protein quantification tool, we developed immuno-matrix-assisted laser desorption/ionization (iMALDI) assays with automated liquid handling. The assays are able to quantify AKT1 and AKT2 from breast cancer and colon cancer cell lines and flash-frozen tumor lysates with a linear range of 0.05-2.0 fmol/µg of total lysate protein and with coefficients of variation < 15%. Compared to other mass spectrometric methods, the developed assays require less sample per analysis-only 25 µg of total protein-and are therefore suitable for analysis of needle biopsies. Furthermore, the presented iMALDI technique is the first MS-based method for absolute quantitation of AKT peptides from cancer tissues. This study demonstrates the suitability of iMALDI for low limit-of-detection and reproducible quantitation of signaling pathway members using a benchtop MALDI mass spectrometer within approximately 6-7 h.

Protocol for Standardizing High-to-Moderate Abundance Protein Biomarker Assessments Through an MRM-with-Standard-Peptides Quantitative Approach.

Quantitative mass spectrometry (MS)-based approaches are emerging as a core technology for addressing health-related queries in systems biology and in the biomedical and clinical fields. In several 'omics disciplines (proteomics included), an approach centered on selected or multiple reaction monitoring (SRM or MRM)-MS with stable isotope-labeled standards (SIS), at the protein or peptide level, has emerged as the most precise technique for quantifying and screening putative analytes in biological samples. To enable the widespread use of MRM-based protein quantitation for disease biomarker assessment studies and its ultimate acceptance for clinical analysis, the technique must be standardized to facilitate precise and accurate protein quantitation. To that end, we have developed a number of kits for assessing method/platform performance, as well as for screening proposed candidate protein biomarkers in various human biofluids. Collectively, these kits utilize a bottom-up LC-MS methodology with SIS peptides as internal standards and quantify proteins using regression analysis of standard curves. This chapter details the methodology used to quantify 192 plasma proteins of high-to-moderate abundance (covers a 6 order of magnitude range from 31 mg/mL for albumin to 18 ng/mL for peroxidredoxin-2), and a 21-protein subset thereof. We also describe the application of this method to patient samples for biomarker discovery and verification studies. Additionally, we introduce our recently developed Qualis-SIS software, which is used to expedite the analysis and assessment of protein quantitation data in control and patient samples.

Increased Depth and Breadth of Plasma Protein Quantitation via Two-Dimensional Liquid Chromatography/Multiple Reaction Monitoring-Mass Spectrometry with Labeled Peptide Standards.

Absolute quantitative strategies are emerging as a powerful and preferable means of deriving concentrations in biological samples for systems biology applications. Method development is driven by the need to establish new-and validate current-protein biomarkers of high-to-low abundance for clinical utility. In this chapter, we describe a methodology involving two-dimensional (2D) reversed-phase liquid chromatography (RPLC), operated under alkaline and acidic pH conditions, combined with multiple reaction monitoring (MRM)-mass spectrometry (MS) (also called selected reaction monitoring (SRM)-MS) and a complex mixture of stable isotope-labeled standard (SIS) peptides, to quantify a broad and diverse panel of 253 proteins in human blood plasma. The quantitation range spans 8 orders of magnitude-from 15 mg/mL (for vitamin D-binding protein) to 450 pg/mL (for protein S100-B)-and includes 31 low-abundance proteins (defined as being <10 ng/mL) of potential disease relevance. The method is designed to assess candidates at the discovery and/or verification phases of the biomarker pipeline and can be adapted to examine smaller or alternate panels of proteins for higher sample throughput. Also detailed here is the application of our recently developed software tool-Qualis-SIS-for protein quantitation (via regression analysis of standard curves) and quality assessment of the resulting data. Overall, this chapter provides the blueprint for the replication of this quantitative proteomic method by proteomic scientists of all skill levels.

Exploring phlebotomy technique as a pre-analytical factor in proteomic analyses by mass spectrometry.

Multiple reaction monitoring mass spectrometry (MRM-MS) is an emerging technology for blood biomarker verification and validation; however, the results may be influenced by pre-analytical factors. This exploratory study was designed to determine if differences in phlebotomy techniques would significantly affect the abundance of plasma proteins in an upcoming biomarker development study. Blood was drawn from 10 healthy participants using four techniques: (1) a 20-gauge IV with vacutainer, (2) a 21-gauge direct vacutainer, (3) an 18-gauge butterfly with vacutainer, and (4) an 18-gauge butterfly with syringe draw. The abundances of a panel of 122 proteins (117 proteins, plus 5 matrix metalloproteinase (MMP) proteins) were targeted by LC/MRM-MS. In addition, complete blood count (CBC) data were also compared across the four techniques. Phlebotomy technique significantly affected 2 of the 11 CBC parameters (red blood cell count, p = 0.010; hemoglobin concentration, p = 0.035) and only 12 of the targeted 117 proteins (p < 0.05). Of the five MMP proteins, only MMP7 was detectable and its concentration was not significantly affected by different techniques. Overall, most proteins in this exploratory study were not significantly influenced by phlebotomy technique; however, a larger study with additional patients will be required for confirmation.

Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots.

The dried blood spot (DBS) methodology provides a minimally invasive approach to sample collection and enables room-temperature storage for most analytes. DBS samples have successfully been analyzed by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS) to quantify a large range of small molecule biomarkers and drugs; however, this strategy has only recently been explored for MS-based proteomics applications. Here we report the development of a highly multiplexed MRM assay to quantify endogenous proteins in human DBS samples. This assay uses matching stable isotope-labeled standard peptides for precise, relative quantification, and standard curves to characterize the analytical performance. A total of 169 peptides, corresponding to 97 proteins, were quantified in the final assay with an average linear dynamic range of 207-fold and an average R(2) value of 0.987. The total range of this assay spanned almost 5 orders of magnitude from serum albumin (P02768) at 18.0 mg/ml down to cholinesterase (P06276) at 190 ng/ml. The average intra-assay and inter-assay precision for 6 biological samples ranged from 6.1-7.5% CV and 9.5-11.0% CV, respectively. The majority of peptide targets were stable after 154 days at storage temperatures from -20 °C to 37 °C. Furthermore, protein concentration ratios between matching DBS and whole blood samples were largely constant (<20% CV) across six biological samples. This assay represents the highest multiplexing yet achieved for targeted protein quantification in DBS samples and is suitable for biomedical research applications.

Precise quantitation of 136 urinary proteins by LC/MRM-MS using stable isotope labeled peptides as internal standards for biomarker discovery and/or verification studies.

Spurred on by the growing demand for panels of validated disease biomarkers, increasing efforts have focused on advancing qualitative and quantitative tools for more highly multiplexed and sensitive analyses of a multitude of analytes in various human biofluids. In quantitative proteomics, evolving strategies involve the use of the targeted multiple reaction monitoring (MRM) mode of mass spectrometry (MS) with stable isotope-labeled standards (SIS) used for internal normalization. Using that preferred approach with non-invasive urine samples, we have systematically advanced and rigorously assessed the methodology toward the precise quantitation of the largest, multiplexed panel of candidate protein biomarkers in human urine to date. The concentrations of the 136 proteins span >5 orders of magnitude (from 8.6 µg/mL to 25 pg/mL), with average CVs of 8.6% over process triplicate. Detailed here is our quantitative method, the analysis strategy, a feasibility application to prostate cancer samples, and a discussion of the utility of this method in translational studies.

An automated assay for the clinical measurement of plasma renin activity by immuno-MALDI (iMALDI).

Plasma renin activity (PRA) is essential for the screening and diagnosis of primary aldosteronism (PA), a form of secondary hypertension, which affects approximately 100 million people worldwide. It is commonly determined by radioimmunoassay (RIA) and, more recently, by relatively low-throughput LC-MS/MS methods. In order to circumvent the negative aspects of RIAs (radioisotopes, cross-reactivity) and the low throughput of LC-MS based methods, we have developed a high-throughput immuno-MALDI (iMALDI)-based assay for PRA determination using an Agilent Bravo for automated liquid handling and a Bruker Microflex LRF instrument for MALDI analysis, with the goal of implementing the assay in clinical laboratories. The current assay allows PRA determination of 29 patient samples (192 immuno-captures), within ~6 to 7h, using a 3-hour Ang I generation period, at a 7.5-fold faster analysis time than LC-MS/MS. The assay is performed on 350µL of plasma, and has a linear range from 0.08 to 5.3ng/L/s in the reflector mode, and 0.04 to 5.3ng/L/s in the linear mode. The analytical precision is 2.0 to 9.7% CV in the reflector mode, and 1.5 to 14.3% CV in the linear mode. A method comparison to a clinically employed LC-MS/MS assay for PRA determination showed excellent correlation within the linear range, with an R(2) value of ≥0.98. This automated high throughput iMALDI platform has clinically suitable sensitivity, precision, linear range, and correlation with the standard method for PRA determination. Furthermore, the developed workflow based on the iMALDI technology can be used for the determination of other proteomic biomarkers. This article is part of a Special Issue entitled: Medical Proteomics.

Qualis-SIS: automated standard curve generation and quality assessment for multiplexed targeted quantitative proteomic experiments with labeled standards.

Multiplexed targeted quantitative proteomics typically utilizes multiple reaction monitoring and allows the optimized quantification of a large number of proteins. One challenge, however, is the large amount of data that needs to be reviewed, analyzed, and interpreted. Different vendors provide software for their instruments, which determine the recorded responses of the heavy and endogenous peptides and perform the response-curve integration. Bringing multiplexed data together and generating standard curves is often an off-line step accomplished, for example, with spreadsheet software. This can be laborious, as it requires determining the concentration levels that meet the required accuracy and precision criteria in an iterative process. We present here a computer program, Qualis-SIS, that generates standard curves from multiplexed MRM experiments and determines analyte concentrations in biological samples. Multiple level-removal algorithms and acceptance criteria for concentration levels are implemented. When used to apply the standard curve to new samples, the software flags each measurement according to its quality. From the user's perspective, the data processing is instantaneous due to the reactivity paradigm used, and the user can download the results of the stepwise calculations for further processing, if necessary. This allows for more consistent data analysis and can dramatically accelerate the downstream data analysis.

Multiplexed MRM with Internal Standards for Cerebrospinal Fluid Candidate Protein Biomarker Quantitation.

Multiplexed quantitation is essential for discovering, verifying, and validating biomarkers for risk stratification, disease prognostication, and therapeutic monitoring. The most promising strategy for quantifying unverified protein biomarkers in biofluids relies on selected/multiple reaction monitoring (SRM or MRM) technology with isotopically labeled standards employed within a bottom-up proteomic workflow. Since cerebrospinal fluid (CSF) is an important fluid for studying central nervous system (CNS) related diseases, we sought to develop a rapid, antibody- and fractionation-free MRM-based approach with a complex mixture of peptide standards to quantify a highly multiplexed panel of candidate protein biomarkers in human CSF. Development involved peptide transition optimization, denaturation/digestion protocol evaluation, transition interference screening, and protein quantitation via peptide standard curves. The final method exhibited excellent reproducibility (average coefficient of variation of <1% for retention time and <6% for signal) and breadth of quantitation (130 proteins from 311 interference-free peptides) in a single 43-min run. These proteins are of high-to-low abundance with determined concentrations from 118 µg/mL (serum albumin) to 550 pg/mL (apolipoprotein C-I). Overall, the method consists of the most highly multiplexed and broadest panel of candidate protein biomarkers in human CSF reported thus far and is well suited for subsequent verification studies on patient samples.

Enhanced sensitivity and multiplexing with 2D LC/MRM-MS and labeled standards for deeper and more comprehensive protein quantitation.

Mass spectrometry (MS)-based protein quantitation is increasingly being employed to verify candidate protein biomarkers. Multiple or selected reaction monitoring-mass spectrometry (MRM-MS or SRM-MS) with isotopically labeled internal standards has proven to be a successful approach in that regard, but has yet to reach its full potential in terms of multiplexing and sensitivity. Here, we report the development of a new MRM method for the quantitation of 253 disease-associated proteins (represented by 625 interference-free peptides) in 13 LC fractions. This 2D RPLC/MRM-MS approach extends the depth and breadth of the assay by 2 orders of magnitude over pre-fractionation-free assays, with 31 proteins below 10 ng/mL and 41 proteins above 10 ng/mL now quantifiable. Standard flow rates are used in both chromatographic dimensions, and up-front depletion or antibody-based enrichment is not required. The LC separations utilize high and low pH conditions, with the former employing an ammonium hydroxide-based eluent, instead of the conventional ammonium formate, resulting in improved LC column lifetime and performance. The high sensitivity (determined concentration range: 15 mg/mL to 452 pg/mL) and robustness afforded by this method makes the full MRM panel, or subsets thereof, useful for the verification of disease-associated plasma protein biomarkers in patient samples. BIOLOGICAL SIGNIFICANCE: The described research extends the breadth and depth of protein quantitation in undepleted and non-enriched human plasma by employing standard-flow 2D RPLC/MRM-MS in conjunction with a complex mixture of isotopically labeled peptide standards. The proteins quantified are mainly putative biomarkers of non-communicable (i.e., non-infectious) disease (e.g., cardiovascular or cancer), which require pre-clinical verification and validation before clinical implementation. Based on the enhanced sensitivity and multiplexing, this quantitative plasma proteomic method should prove useful in future candidate biomarker verification studies.

MRM for the verification of cancer biomarker proteins: recent applications to human plasma and serum.

Accurate cancer biomarkers are needed for early detection, disease classification, prediction of therapeutic response and monitoring treatment. While there appears to be no shortage of candidate biomarker proteins, a major bottleneck in the biomarker pipeline continues to be their verification by enzyme linked immunosorbent assays. Multiple reaction monitoring (MRM), also known as selected reaction monitoring, is a targeted mass spectrometry approach to protein quantitation and is emerging to bridge the gap between biomarker discovery and clinical validation. Highly multiplexed MRM assays are readily configured and enable simultaneous verification of large numbers of candidates facilitating the development of biomarker panels which can increase specificity. This review focuses on recent applications of MRM to the analysis of plasma and serum from cancer patients for biomarker verification. The current status of this approach is discussed along with future directions for targeted mass spectrometry in clinical biomarker validation.

Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility.

Accurate and rapid protein quantitation is essential for screening biomarkers for disease stratification and monitoring, and to validate the hundreds of putative markers in human biofluids, including blood plasma. An analytical method that utilizes stable isotope-labeled standard (SIS) peptides and selected/multiple reaction monitoring-mass spectrometry (SRM/MRM-MS) has emerged as a promising technique for determining protein concentrations. This targeted approach has analytical merit, but its true potential (in terms of sensitivity and multiplexing) has yet to be realized. Described herein is a method that extends the multiplexing ability of the MRM method to enable the quantitation 142 high-to-moderate abundance proteins (from 31mg/mL to 44ng/mL) in undepleted and non-enriched human plasma in a single run. The proteins have been reported to be associated to a wide variety of non-communicable diseases (NCDs), from cardiovascular disease (CVD) to diabetes. The concentrations of these proteins in human plasma are inferred from interference-free peptides functioning as molecular surrogates (2 peptides per protein, on average). A revised data analysis strategy, involving the linear regression equation of normal control plasma, has been instituted to enable the facile application to patient samples, as demonstrated in separate nutrigenomics and CVD studies. The exceptional robustness of the LC/MS platform and the quantitative method, as well as its high throughput, makes the assay suitable for application to patient samples for the verification of a condensed or complete protein panel. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Mass spectrometry in high-throughput clinical biomarker assays: multiple reaction monitoring.

Clinical biomarker discovery, verification, and validation are facilitated by the latest technological advances in mass spectrometry. It is now possible to analyze simultaneously group of tens or hundreds of biomarkers in a blood sample using multiple reaction monitoring (MRM), a tandem mass spectrometric method. However, these newly-developed methods face new challenges, including standardization, calibration, and the determination of analytical and biological variation. Here we illustrate the background, pre-analytical sample preparation, and biomarker assay development using an MRM-mass spectrometric method. In addition, special attention is given to future standardization methods to enable widespread use of the technology.

Targeted quantitation of CVD-linked plasma proteins for biomarker verification and validation.

Despite significant advances in treatment, cardiovascular disease (CVD) remains one of the leading causes of morbidity and mortality in developed and developing countries. Judicious monitoring of common risk factors has been unable to control this global epidemic, necessitating novel biomarkers for improved screening and earlier disease detection and management. Although numerous plasma proteins have been associated with CVD, only a few of these potential biomarkers have been validated for clinical use. Here we review the quantitative proteomic methods used to verify and validate new biomarker candidates in human plasma. These methods center on a bottom-up approach involving multiple or selected reaction monitoring, for targeted detection, with stable isotope-labeled standards, for peptide normalization. Also included are a discussion of future strategies for improved CVD protein biomarker verification and validation, recommendations for method translation to the clinic, and future projections for protein biomarker research.

Method and platform standardization in MRM-based quantitative plasma proteomics.

There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. BIOLOGICAL SIGNIFICANCE: The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.

Comparison of proteins in whole blood and dried blood spot samples by LC/MS/MS.

Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77%-92% within replicates and the majority of these repeated proteins (70%) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.