Stable Isotope-labeled Standard Peptides Research Articles

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

Codon optimization based on psbA genes from 133 plant species eliminated 105 (human clotting factor VIII heavy chain [FVIII HC]) and 59 (polio VIRAL CAPSID PROTEIN1 [VP1]) rare codons; replacement with only the most highly preferred codons decreased transgene expression (77- to 111-fold) when compared with the codon usage hierarchy of the psbA genes. Targeted proteomic quantification by parallel reaction monitoring analysis showed 4.9- to 7.1-fold or 22.5- to 28.1-fold increase in FVIII or VP1 codon-optimized genes when normalized with stable isotope-labeled standard peptides (or housekeeping protein peptides), but quantitation using western blots showed 6.3- to 8-fold or 91- to 125-fold increase of transgene expression from the same batch of materials, due to limitations in quantitative protein transfer, denaturation, solubility, or stability. Parallel reaction monitoring, to our knowledge validated here for the first time for in planta quantitation of biopharmaceuticals, is especially useful for insoluble or multimeric proteins required for oral drug delivery. Northern blots confirmed that the increase of codon-optimized protein synthesis is at the translational level rather than any impact on transcript abundance. Ribosome footprints did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but is useful in diagnosing additional rate-limiting steps. A major ribosome pause at CTC leucine codons in the native gene of FVIII HC was eliminated upon codon optimization. Ribosome stalls observed at clusters of serine codons in the codon-optimized VP1 gene provide an opportunity for further optimization. In addition to increasing our understanding of chloroplast translation, these new tools should help to advance this concept toward human clinical studies.

Optimized Protocol for Quantitative Multiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues.

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R(2) = 0.94) and immuno-MRM (R(2) = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.

Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry

The BRAF V600E mutation is one of the most common mutations implicated in the development of several types of cancer including colorectal cancer (CRC), where it is associated with aggressive disease phenotypes and poor outcomes. The status of the BRAF V600E mutation is frequently determined by direct DNA sequencing. However, no previous study has sought to quantify the BRAF V600E protein in cancer specimens. Here, we evaluated immunoenrichment coupled with two MS-based quantitative techniques, namely multiple reaction monitoring (MRM) and single ion monitoring conjugated accurate inclusion mass screening (SIM-AIMS), to detect and precisely quantify wild-type (WT) and V600E mutant BRAF proteins in DNA sequence-confirmed CRC tissue specimens. WT and V600E BRAF proteins were immunoprecipitated from a CRC cell line (HT-29), and their representative peptides (592IGDFGLATVK601 and 592IGDFGLATEK601, respectively) were confirmed by LC-MS/MS analysis and then quantified by MRM or SIM-AIMS with spiked stable isotope-labeled peptide standards. Both assays worked well for measuring WT BRAF from different amounts of HT-29 cell lysates, but the MRM assay was more sensitive than SIM-AIMS assay for quantifying lower levels of V600E BRAF. In protein extracts (2 mg) from 11 CRC tissue specimens, the MRM assay could measure WT BRAF in all 11 cases (0.32–1.66 ng) and the V600E BRAF in two cases (0.1–0.13 ng; mutant-to-WT ratio, 0.16–0.17). The SIM-AIMS assay could also detect WT and V600E BRAF in CRC specimens, but the measured levels of both targets were lower than those determined by MRM assay. Collectively, this study provides an effective method to precisely quantify WT and V600E BRAF proteins in complex biological samples using immunoenrichment-coupled targeted MS. Since the V600E BRAF protein has emerged as an important therapeutic target for cancer, the developed assay should facilitate future BRAF-related basic and clinical studies.

Abstract 538: Metabolism of Multiple Apolipoproteins Across HDL Size in Humans

Introduction: Studying the kinetics of several HDL apolipoproteins together may help elucidate their function in HDL metabolism. We present multicompartmental analysis describing the metabolism of several apolipoproteins across 5 HDL size fractions. Methods and Results: Three participants were infused with a bolus of D3-Leu tracer, and blood was collected for 70 hrs. ApoA-I-HDL was prepared by immunoaffinity purification, separated into 5 size fractions, preβ, α3, α2, α1, and α0, by ND-PAGE, and in-gel trypsinized for mass spectrometry. We monitored 7 proteins that likely affect HDL metabolism - apoA-I, apoA-II, apoE, apoM, apoC-III, apoA-IV, and apoD. We used stable isotope labeled peptide standards to quantify pool size and high resolution parallel reaction monitoring to measure tracer enrichment in these 7 proteins in the 5 HDL size fractions. These data were then used for multicompartmental analysis to describe the kinetic behaviors across HDL size for each apolipoprotein, with the exception of apoD. All size fractions with discernible enrichment curves for at least 2 participants were included in the final model for each apolipoprotein (Fig.1). The majority of apoA-I α HDL originated form the source compartment, presumably the liver and/or small intestine, while apoA-I preβ HDL came primarily from α3. Size expansion pathways (preβ to α1/2, and α3 to α2) contributed only slightly to apoA-I metabolism. Similarly to apoA-I on the α sizes, the majority of the other apolipoproteins appear on each HDL size fraction directly from the source compartment. Only minor flux pathways from smaller to larger HDL sizes were identified: apoA-II, α3 to α2; apoE, α3 to α2, and α3 to α1. Conclusions: This study supports the new model of HDL metabolism in which HDL and its attached apolipoproteins are metabolized mainly within each HDL size fraction. Pathways between HDL sizes only provide a small contribution to the metabolism of each apolipoprotein.

Multiplexed Immunoaffinity Enrichment of Peptides with Anti-peptide Antibodies and Quantification by Stable Isotope Dilution Multiple Reaction Monitoring Mass Spectrometry.

Immunoaffinity enrichment of peptides using anti-peptide antibodies and their subsequent analysis by targeted mass spectrometry using stable isotope-labeled peptide standards is a sensitive and relatively high-throughput assay technology for unmodified and modified peptides in cells, tissues, and biofluids. Suppliers of antibodies and peptides are increasingly aware of this technique and have started incorporating customized quality measures and production protocols to increase the success rate, performance, and supply of the necessary reagents. Over the past decade, analytical biochemists, clinical diagnosticians, antibody experts, and mass spectrometry specialists have shared ideas, instrumentation, reagents, and protocols, to demonstrate that immuno-MRM-MS is reproducible across laboratories. Assay performance is now suitable for verification of candidate biomarkers from large scale discovery "omics" studies, measuring diagnostic proteins in plasma in the clinical laboratory, and for developing a companion assay for preclinical drug studies. Here we illustrate the process for developing these assays with a step-by-step guide for a 20-plex immuno-MRM-MS assay. We emphasize the need for analytical validation of the assay to insure that antibodies, peptides, and mass spectrometer are working as intended, in a multiplexed manner, with suitable assay performance (median values for 20 peptides: CV = 12.4% at 740 amol/μL, LOD = 310 amol/μL) for applications in quantitative biology and candidate biomarker verification. The assays described conform to Tier 2 (of 3) level of analytical assay validation (1), meaning that the assays are capable of repeatedly measuring sets of analytes of interest within and across samples/experiments and employ internal standards for each analyte for confident detection and precise quantification.

Monitoring PPARG-Induced Changes in Glycolysis by Selected Reaction Monitoring Mass Spectrometry.

As cells develop and differentiate, they change in function and morphology, which often precede earlier changes in signaling and metabolic control. Here we present a selected reaction monitoring (SRM) approach which allows for the parallel quantification of metabolic regulators and their downstream targets.In particular we explain and describe how to monitor abundance changes of glycolytic enzymes upon PPARγ activation by using a label-free or a stable isotope-labeled standard peptide (SIS peptides) approach applying triple-quadrupole mass spectrometry. We further outline how to fractionate the cell lysate into cytosolic and nuclear fractions to enhance the sensitivity of the measurements and to investigate the dynamic concentration changes in those compartments.

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.

An integrated quantification method to increase the precision, robustness, and resolution of protein measurement in human plasma samples.

BackgroundCurrent quantification methods for mass spectrometry (MS)-based proteomics either do not provide sufficient control of variability or are difficult to implement for routine clinical testing.ResultsWe present here an integrated quantification (InteQuan) method that better controls pre-analytical and analytical variability than the popular quantification method using stable isotope-labeled standard peptides (SISQuan). We quantified 16 lung cancer biomarker candidates in human plasma samples in three assessment studies, using immunoaffinity depletion coupled with multiple reaction monitoring (MRM) MS. InteQuan outperformed SISQuan in precision in all three studies and tolerated a two-fold difference in sample loading. The three studies lasted over six months and encountered major changes in experimental settings. Nevertheless, plasma proteins in low ng/ml to low μg/ml concentrations were measured with a median technical coefficient of variation (CV) of 11.9% using InteQuan. The corresponding median CV using SISQuan was 15.3% after linear fitting. Furthermore, InteQuan surpassed SISQuan in measuring biological difference among clinical samples and in distinguishing benign versus cancer plasma samples.ConclusionsWe demonstrated that InteQuan is a simple yet robust quantification method for MS-based quantitative proteomics, especially for applications in biomarker research and in routine clinical testing.Electronic supplementary materialThe online version of this article (doi:10.1186/1559-0275-12-3) contains supplementary material, which is available to authorized users.

Evaluation of Interspecimen Trypsin Digestion Efficiency Prior to Multiple Reaction Monitoring-Based Absolute Protein Quantification with Native Protein Calibrators

Implementation of quantitative clinical chemistry proteomics (qCCP) requires targeted proteomics approaches, usually involving bottom-up multiple reaction monitoring-mass spectrometry (MRM-MS) with stable-isotope labeled standard (SIS) peptides, to move toward more accurate measurements. Two aspects of qCCP that deserve special attention are (1) proper calibration and (2) the assurance of consistent digestion. Here, we describe the evaluation of tryptic digestion efficiency by monitoring various signature peptides, missed cleavages, and modifications during proteolysis of apolipoprotein A-I and B in normo- and hypertriglyceridemic specimens. Absolute quantification of apolipoprotein A-I and B was performed by LC-MRM-MS with SIS peptide internal standards at two time points (4 and 20 h), using three native protein calibrators. Comparison with an immunoturbidimetric assay revealed recoveries of 99.4 ± 6.5% for apolipoprotein A-I and 102.6 ± 7.2% for apolipoprotein B after 4 h of trypsin digestion. Protein recoveries after 20 h trypsin incubation equaled 95.9 ± 6.9% and 106.0 ± 10.0% for apolipoproteins A-I and B, respectively. In conclusion, the use of metrologically traceable, native protein calibrators looks promising for accurate quantification of apolipoprotein A-I and B. Selection of rapidly formed peptides, that is, with no or minor missed cleavages, and the use of short trypsin incubation times for these efficiently cleaved peptides are likely to further reduce the variability introduced by trypsin digestion and to improve the traceability of test results to reach the desirable analytical performance for clinical chemistry application.

Targeted Precise Quantification of 12 Human Recombinant Uridine-Diphosphate Glucuronosyl Transferase 1A and 2B Isoforms Using Nano-Ultra-High-Performance Liquid Chromatography/Tandem Mass Spectrometry with Selected Reaction Monitoring

Quantification methods employing stable isotope-labeled peptide standards and liquid chromatography-tandem mass spectrometry are increasingly being used to measure enzyme amounts in biologic samples. Isoform concentrations, combined with catalytic information, can be used in absorption, distribution, metabolism, and excretion studies to improve accuracy of in vitro/in vivo predictions. We quantified isoforms of uridine-diphosphate glucuronosyltransferase (UGT) 1A and 2B in 12 commercially available recombinant UGTs (recUGTs) (n = 49 samples) using nano-ultra-high-performance liquid chromatography-tandem mass spectrometry with selected reaction monitoring). Samples were trypsin-digested and analyzed using our previously published method. Two MRMs were collected per peptide and averaged. Where available, at least two peptides were measured per UGT isoform. The assay could detect UGTs in all recombinant preparations: recUGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17, with limit of detection below 1.0 pmol/mg protein for all isoforms. The assay had excellent linearity in the range observed (2-15.5 pmol/mg, after dilution). Examples of concentrations determined were 1465, 537, 538, 944, 865, 698, 604, 791, 382, 1149, 307, and 740 pmol/mg protein for the respective isoforms. There was a 6.9-fold difference between the maximum and minimum recUGT concentrations. The range of concentrations determined indicates that catalytic rates per mg total protein in vitro will not accurately reflect isoform inherent specific activity for a particular drug candidate. This is the first report of a targeted precise quantification of commercially available recUGTs. The assay has potential for use in comparing UGT amounts with catalytic activity determined using probe substrates, thus allowing representation of catalysis as per pmol of UGT isoform.

Multiple Reaction Monitoring of Multiple Low-Abundance Transcription Factors in Whole Lung Cancer Cell Lysates

Lung cancer-related transcription factors (TFs) were identified by integrating previously reported genomic, transcriptomic, and proteomic data and were quantified by multiple reaction monitoring (MRM) in various cell lines. All experiments were performed without affinity depletion or subfractionation of cell lysates. Since the target proteins were expected to be present in low abundance, we experimentally optimized MRM transition parameters with chemically synthesized peptides. Quantitation was based on stable isotope-labeled standard peptides (SIS peptides). Out of 288 MRM measurements (36 peptides representing 28 TFs × 8 cell lines), 241 were successfully obtained within a quantitation limit of 15 amol, 221 measurements (91.7%) showed coefficients of variation (CVs) of ≤ 20%, and 149 (61.8%) showed CVs of ≤ 10%, quantifying as low as 19.4 amol/μg protein for STAT2 with a CV of 6.3% in an A549 cell. Comparisons between MRM measurements and levels of the corresponding mRNAs revealed linear, nonlinear, or no relationship between protein and mRNA levels, indicating the need for an MRM assay. An integrative analysis of MRM and gene expression profiles from doxorubicin-resistant H69AR and sensitive H69 cells further showed that 14 differentially expressed TFs, such as STAT1 and SMAD4, regulated genes associated with drug resistance and cell differentiation-related processes. Thus, the analytical performance of MRM for the quantitation of low abundance TFs suggests its usefulness for biological application.

Abstract 2494: Liquid chromatography-multiple reaction monitoring mass spectrometry biomarker quantification for breast cancer patient assessment.

Abstract Introduction: A quantitative mass spectrometry platform has been developed for measuring biomarkers of protein expression and phosphorylation in breast cancer. The quantification of numerous biomarkers from a single tissue specimen has the potential to impact patient care. Specifically, the focus of initial development was on the expression and phosphorylation of estrogen receptor alpha (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). However, immunohistochemical assessments of Ki-67 and vimentin are also widely used for monitoring proliferation in breast cancer. The integration of tissue quality control measurements, quantification of proliferation biomarkers, and ER/PR/HER2 evaluation could improve patient assessment and open novel avenues for patient classification, prognosis, and selection of therapy in breast cancer. Experimental Procedures: Breast cancer cell lines MCF7, BT474, and T47D were used to develop peptide-based quantitative assays, including stable isotope-labeled standard peptides, (n = 31) for protein expression and phosphorylation biomarkers using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) of digested whole cell lysates or SDS-PAGE fractionated proteins followed by immunoprecipitation to examine expression and phosphorylation of ER/PR/HER2 (IP-LC-MRM). Using 2 mg of total protein, the sample is divided into three parts. Tissue QC biomarkers and expression levels of Ki67, vimentin, and HER2 are measured in digests of whole cell lysate or tissue homogenate (1% of sample). Then, parallel IP strategies are used for HER2 (10% of sample) and ER/PR (90%) before LC-MRM analysis of expression and phosphorylation. Multiplex IP of the three proteins was not successful, because the amplification of HER2 could not be accommodated. The sensitivity of each assay was tested using serial dilutions of cell lysate to evaluate requirements for analysis of clinical specimens. Data Summary: LC-MRM assays for both unmodified and phosphorylated peptides have been developed and characterized for each protein using cell line models. Assays have been implemented in cell lines to study Estradiol/EGF stimulation as well as lapatinib inhibition of HER2. Data has been generated for pre-treatment frozen tissue specimens from breast cancer patients that are either HER2+ or ER+ (n = 12 per group). The analysis of a larger group of patients and in clinical scenarios (e.g. comparison of naïve and drug resistant tissues from the same patient) will establish the clinical utility of these assays. Conclusions: The ability to examine protein expression as well as phosphorylation status in these biomarkers with quantitative mass spectrometry has the potential to improve the selection of targeted therapeutics. Citation Format: Yi Chen, David J. Britton, Elizabeth R. Wood, Anthony M. Magliocco, Ian Pike, John M. Koomen. Liquid chromatography-multiple reaction monitoring mass spectrometry biomarker quantification for breast cancer patient assessment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2494. doi:10.1158/1538-7445.AM2013-2494

Abstract 4810: Multiplexed immunoprecipitation-liquid chromatography-multiple reaction monitoring mass spectrometry quantification of protein expression and phosphorylation for breast cancer biomarkers

Abstract Introduction: Estrogen receptor alpha (ER alpha), progesterone receptor B (PR B), and the receptor tyrosine kinase, Her2, are tumor markers classified by the American Society of Clinical Oncology (ASCO) as level of evidence I in breast cancer. The quantitative evaluation of protein expression and phosphorylation status of these targets in tumor tissues will enable subtype classification and support clinical decision-making and selection of targeted therapy in breast cancer. Experimental Procedures: Breast cancer cell lines were used to develop peptide-based quantitative assays for protein expression and phosphorylation using immunoprecipitation (IP) and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM). Stable isotope-labeled standard peptides were synthesized and developed as internal standards. Multiplexed IP for ER alpha, PR B, and HER2 will be developed to enable the enrichment of the proteins to enable quantification of the expression and phosphorylation of these biomarkers in the same sample. The sensitivity of each assay will be tested using serial dilutions of cell lysate to evaluate requirements for analysis of clinical specimens. Data Summary: LC-MRM assays for both unmodified and phosphorylated peptides have been developed and characterized for each protein. Recovery using multiplexed IP for these proteins is similar to recovery of individual IP. Data have been generated for cell line models of stimulation and treatment. For example, LC-MRM quantification of HER2 in cell line models stimulated with EGF or Heregulin shows that protein expression is unchanged, but phosphorylation increases; on the other hand, LC-MRM shows clearly that phosphorylation of Her2 is inhibited with Lapatinib. Conclusions: The ability to examine the protein expression as well as the phosphorylation status in these biomarkers with quantitative mass spectrometry will produce improved understanding of their role in the development and progression of breast cancer and has the potential to improve the selection of targeted therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4810. doi:1538-7445.AM2012-4810

MRM‐based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma

A highly-multiplexed MRM-based assay for determination of cardiovascular disease (CVD) status and disease classification has been developed for clinical research. A high-flow system using ultra-high performance LC and an Agilent 6490 triple quadrupole mass spectrometer, equipped with an ion funnel, provided ease of use and increased the robustness of the assay. The assay uses 135 stable isotope-labeled peptide standards for the quantitation of 67 putative biomarkers of CVD in tryptic digests of whole plasma in a 30-min assay. Eighty-five analyses of the same sample showed no loss of sensitivity (<20% CV for 134/135 peptides) and no loss of retention time accuracy (<0.5% CV for all peptides). The maximum linear dynamic range of the MRM assays ranged from 10(3) -10(5) for 106 of the assays. Excellent linear responses (r >0.98) were obtained for 117 of the 135 peptide targets with attomole level limits of quantitation (<20% CV and accuracy 80-120%) for 81 of the 135 peptides. The assay presented in this study is easy to use, robust, sensitive, and has high-throughput capabilities through short analysis time and complete automated sample preparation. It is therefore well suited for CVD biomarker validation and discovery in plasma.

Abstract 17763: Multiple Reaction Monitoring-Based, Multiplexed, Absolute Quantitation of 82 Putative Biomarkers of Cardiovascular Disease in Human Plasma

Multiplexed biomarker expression profiling of clinical samples can be achieved in a rapid and targeted fashion using mass spectrometry-based multiple reaction monitoring (MRM) quantitation. For this project, a mixture of ~230 peptides standards was created to permit absolute quantitation of 82 putative biomarkers of cardiovascular disease, in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Synthetic purified stable isotope-labeled standard peptides (SIS peptides) were added to the samples after tryptic digestion. For maximum specificity, a high-flow system using UPLC and an Agilent 6490 triple quadrupole mass spectrometer, equipped with an ion-funnel sprayer, were used. Instrumental parameters were empirically determined in order to generate the most abundant precursor ions and ion fragments. The narrow UPLC peak shapes and reproducible retention times assisted in the development of the highly-multiplexed system by increasing the chromatographic “space”. In addition, the larger id column, with a larger amount of packing material, also provided increased “robustness” of the overall system, as demonstrated by 110 analyses of the same sample with no loss of sensitivity or retention time accuracy. Linear responses and lowest limits of quantitation were obtained for all proteins. Sensitivities using the new system were in the low attomole range demonstrating that the high-flow system, which allowed loading more protein digest onto the column, more than compensated for any reduction in electrospray ionization efficiency caused by the higher flow rate. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes for the highest quantitation accuracy. The analytical precision was assessed for each protein assay from LC-MRM/MS analyses performed on 3 different days on different batches of plasma tryptic digests, and was found to be within 10%. Concentrations of proteins were compared to reported literature values. This method, with the same mixture of internal standards, is now being used for the plasma protein expression profiling of cardiovascular disease patients.

Analysis of altered protein trafficking in schizophrenia by targeted LC‐SRM/MS

N‐methyl‐d‐aspartate (NMDA) receptor hypofunction is currently a leading hypothesis for the etiology of schizophrenia. Recently, our lab directly observed decreased NMDA receptor activity in postmortem prefrontal cortex tissues of patients and linked this dysfunction to altered protein‐protein interactions at the receptor. The NMDA receptor complex is composed of an estimated 100 proteins and is localized to the post‐synaptic density, a thickening of the cell wall at the dendrite rich in receptors, scaffolding proteins and signaling molecules. We hypothesize that alterations to trafficking of the receptor and members of the receptor complex contribute to this dysfunction. To test this hypothesis we have developed a liquid chromatography – selected reaction monitoring/mass spectrometry (LC‐SRM/MS) method utilizing both synthetic stable isotope labeled peptide standards and a surrogate Stable Isotope Labeled Proteome (SILAP) derived from mouse brain tissue labeled with L‐Lysine (13C6). We have used this method to quantify 30 target proteins across several sub‐cellular fractionations of human postmortem pre‐frontal cortex from schizophrenic and matched control subjects. To date we have identified several scaffolding proteins with altered trafficking. We are currently investigating an additional 150 proteins.

Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma

Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([(13)C(6)]Arg or [(13)C(6)]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.

Absolute Quantification of Phosphorylation on the Kinase Activation Loop of Cellular Focal Adhesion Kinase by Stable Isotope Dilution Liquid Chromatography/Mass Spectrometry

A vital point of convergence for many signaling pathways at cellular focal adhesions is the interaction of two nonreceptor tyrosine kinases, focal adhesion kinase (FAK) and Src. The binding of Src to FAK leads to the phosphorylation of Y(576) and Y(577), located within the activation loop domain of FAK. However, it has not been possible previously to determine the absolute quantitative relationship between phosphorylated and nonphosphorylated forms of this activation loop domain in cells undergoing normal metabolism. We have developed a stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) technique that allows such determinations to be made. Isotopically labeled and phosphorylated FAK protein standards were synthesized and used to control for loss during immunoprecipitation of FAK. A control tryptic peptide, representing an unmodified region of FAK, was employed to monitor the mass balance of post-translational modifications (PTMs) on the activation loop domain. Absolute quantification was conducted using stable isotope labeled peptide standards with four endogenous amino acid overhangs at the trypsin digestion sites of both the amino and carboxy terminus. The LC-MRM/MS method was rigorously validated using in vitro kinase assays and employed to conduct absolute quantification of FAK phosphorylation in normal mouse embryonic fibroblasts (MEFs). This methodology will have particular utility for biomarker studies of kinase-inhibiting anticancer drugs and for quantitative proteomic investigations that examine kinase- and phosphatase-mediated cellular signal transduction pathways.

Quantitative Urinary Proteome Analysis for Biomarker Evaluation in Chronic Kidney Disease

A limitation of proteomic methods with respect to their clinical applicability is the lack of possibilities to directly deduce the amount of a protein or peptide from a particular mass spectrometry (MS) spectrum. For quantification of chronic kidney disease (CKD)-specific urinary polypeptides in capillary electrophoresis coupled with mass spectrometry (CE-MS), we compared signal intensity calibration methods based on either urinary creatinine or stable isotope labeled synthetic marker analogues (absolute quantification) with those based on ion counting using highly abundant collagen fragments as nonmarker references (relative quantification). Our results indicate that relative quantification of biomarker excretion based on ion counts in reference to endogenous "housekeeping" peptides is sufficient for the determination of urinary polypeptide levels. The calculation of absolute concentrations via exogenous stable isotope-labeled peptide standards is of no additional benefit.

A multiple reaction monitoring method for absolute quantification of the human liver alcohol dehydrogenase ADH1C1 isoenzyme

Although significant progress has been made in protein quantification using mass spectrometry during recent years, absolute protein quantification in complex biological systems remains a challenging task in proteomics. The use of stable isotope-labeled standard peptide is the most commonly used strategy for absolute quantification, but it might not be suitable in all instances. Here we report an alternative strategy that employs a stable isotope-labeled intact protein as an internal standard to absolutely quantify the alcohol dehydrogenase (ADH) expression level in a human liver sample. In combination with a new targeted proteomics approach employing the method of multiple reaction monitoring (MRM), we precisely and quantitatively measured the absolute protein expression level of an ADH isoenzyme, ADH1C1, in human liver. Isotope-labeled protein standards are predicted to be particularly useful for measurement of highly homologous isoenzymes such as ADHs where multiple signature peptides can be examined by MRM in a single experiment.