Scott Peterman

Increased selectivity, analytical precision, and throughput in targeted proteomics

Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies.

Expediting the Development of Targeted SRM Assays: Using Data from Shotgun Proteomics to Automate Method Development

Selected reaction monitoring (SRM) is a powerful tandem mass spectrometry method that can be used to monitor target peptides within a complex protein digest. The specificity and sensitivity of the approach, as well as its capability to multiplex the measurement of many analytes in parallel, has made it a technology of particular promise for hypothesis driven proteomics. An underappreciated step in the development of an assay to measure many peptides in parallel is the time and effort necessary to establish a usable assay. Here we report the use of shotgun proteomics data to expedite the selection of SRM transitions for target peptides of interest. The use of tandem mass spectrometry data acquired on an LTQ ion trap mass spectrometer can accurately predict which fragment ions will produce the greatest signal in an SRM assay using a triple quadrupole mass spectrometer. Furthermore, we present a scoring routine that can compare the targeted SRM chromatogram data with an MS/MS spectrum acquired by data-dependent acquisition and stored in a library. This scoring routine is invaluable in determining which signal in the chromatogram from a complex mixture best represents the target peptide. These algorithmic developments have been implemented in a software package that is available from the authors upon request.

Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum

OBJECTIVES The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimers, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.

Mass Spectrometric Discovery and Selective Reaction Monitoring (SRM) of Putative Protein Biomarker Candidates in First Trimester Trisomy 21 Maternal Serum

The accurate diagnosis of Trisomy 21 requires invasive procedures that carry a risk of miscarriage. The current state-of-the-art maternal serum screening tests measure levels of PAPP-A, free bhCG, AFP, and uE3 in various combinations with a maximum sensitivity of 60-75% and a false positive rate of 5%. There is currently an unmet need for noninvasive screening tests with high selectivity that can detect pregnancies at risk, preferably within the first trimester. The aim of this study was to apply proteomics and mass spectrometry techniques for the discovery of new putative biomarkers for Trisomy 21 in first trimester maternal serum coupled with the immediate development of quantitative selective reaction monitoring (SRM) assays. The results of the novel workflow were 2-fold: (1) we identified a list of differentially expressed proteins in Trisomy 21 vs Normal samples, including PAPP-A, and (2) we developed a multiplexed, high-throughput SRM assay for verification of 12 new putative markers identified in the discovery experiments. To narrow down the initial large list of differentially expressed candidates resulting from the discovery experiments, we incorporated receiver operating characteristic (ROC) curve algorithms early in the data analysis process. We believe this approach provides a substantial advantage in sifting through the large and complex data typically obtained from discovery experiments. The workflow efficiently mined information derived from high-resolution LC-MS/MS discovery data for the seamless construction of rapid, targeted assays that were performed on unfractionated serum digests. The SRM assay lower limit of detection (LLOD) for the target peptides in a background of digested serum matrix was approximately 250-500 attomoles on column and the limit of accurate quantitation (LOQ) was approximately 1-5 femtomoles on column. The assay error as determined by coefficient of variation at LOQ and above ranged from 0 to 16%. The workflow developed in this study bridges the gap between proteomic biomarker discovery and translation into a clinical research environment. Specifically, for Trisomy 21, the described multiplexed SRM assay provides a vehicle for high-throughput verification of these, and potentially other, peptide candidates on larger sample cohorts.

Measurement of protein phosphorylation stoichiometry by selected reaction monitoring mass spectrometry.

The stoichiometry of protein phosphorylation at specific amino acid sites may be used to infer on the significance of the modification, and its biological function in the cell. However, detection and quantification of phosphorylation stoichiometry in tissue remain a significant challenge. Here we describe a strategy for highly sensitive, label-free quantification of protein phosphorylation stoichiometry. Method development included the analysis of synthetic peptides in order to determine constants to relate the mass spectrometry signals of cognate peptide/phosphopeptide pairs, and the detection of the cognate peptides by using high resolution Fourier Transform mass spectrometry (FTMS) and selected reaction monitoring mass spectrometry (SRM). By analyzing extracted ion currents by FTMS, the phosphorylation stoichiometries of two tyrosine residues (tyrosine-194 and tyrosine-397) in the protein tyrosine kinase Lyn were determined in transfected human HEK293T cells and two cultured human multiple myeloma strains. To achieve high sensitivity to measure phosphorylation stoichiometry in tissue, SRM methods were developed and applied for the analysis of phosphorylation stoichiometries of Lyn phospho-sites in multiple myeloma xenograft tumors. Western immuno-blotting was used to verify mass spectrometry findings. The SRM method has potential applications in analyzing clinical samples wherein protein phosphorylation stoichiometries may represent important pharmacodynamic biomarkers.

Epidermal Growth Factor Receptor Phosphorylation Sites Ser991 and Tyr998 Are Implicated in the Regulation of Receptor Endocytosis and Phosphorylations at Ser1039 and Thr1041

Aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) have causal roles in many human cancers, and post-translational modifications including phosphorylation and ubiquitination and protein-protein interactions directly modulate EGFR function. Quantitative mass spectrometric analyses including selected reaction monitoring (also known as multiple reaction monitoring) were applied to the EGFR and associated proteins. In response to epidermal growth factor (EGF) stimulation of cells, phosphorylations at EGFR Ser991 and Tyr998 accumulated more slowly than at receptor sites involved in RAS-ERK signaling. Phosphorylation-deficient mutant receptors S991A and Y998F activated ERK in response to EGF but were impaired for receptor endocytosis. Consistent with these results, the mutant receptors retained a network of interactions with known signaling proteins including EGF-stimulated binding to the adaptor GRB2. Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin. The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser1039 and Thr1041. These residues reside in a serine/threonine-rich region of the receptor previously implicated in p38 mitogen-activated protein kinase-dependent stress/cytokine-induced EGFR internalization and recycling (Zwang, Y., and Yarden, Y. (2006) p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J. 25, 4195–4206). EGF-induced phosphorylations at Ser1039 and Thr1041 were blocked by treatment of cells with SB-202190, a selective inhibitor of p38. These results suggest that coordinated phosphorylation of EGFR involving sites Tyr998, Ser991, Ser1039, and Thr1041 governs the trafficking of EGF receptors. This reinforces the notion that EGFR function is manifest through spatially and temporally controlled protein-protein interactions and phosphorylations.

Highly multiplexed targeted proteomics using precise control of peptide retention time

Large‐scale proteomics applications using SRM analysis on triple quadrupole mass spectrometers present new challenges to LC‐MS/MS experimental design. Despite the automation of building large‐scale LC‐SRM methods, the increased numbers of targeted peptides can compromise the balance between sensitivity and selectivity. To facilitate large target numbers, time‐scheduled SRM transition acquisition is performed. Previously published results have demonstrated incorporation of a well‐characterized set of synthetic peptides enabled chromatographic characterization of the elution profile for most endogenous peptides. We have extended this application of peptide trainer kits to not only build SRM methods but to facilitate real‐time elution profile characterization that enables automated adjustment of the scheduled detection windows. Incorporation of dynamic retention time adjustments better facilitate targeted assays lasting several days without the need for constant supervision. This paper provides an overview of how the dynamic retention correction approach identifies and corrects for commonly observed LC variations. This adjustment dramatically improves robustness in targeted discovery experiments as well as routine quantification experiments.

An automated, high-throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA-HR/AM)

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC‐MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi‐automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.

Targeted Selected Reaction Monitoring Mass Spectrometric Immunoassay for Insulin-like Growth Factor 1

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.

Targeted phosphoproteomics of insulin signaling using data-independent acquisition mass spectrometry.

A mass spectrometry method may enable the simultaneous and rapid quantification of multiple phosphorylation events in multiple samples. Cross sample proteomics Nontargeted proteomic approaches are not well suited for comparisons of different samples and have detection limits, meaning that many biologically important but low abundance proteins are not detected. By applying a proteomic approach called DIA-MS (data-independent acquisition mass spectrometry) to an adipocyte cell line, Parker et al. showed that this method successfully mapped the phosphorylation events that occurred in response to insulin in a quantitative manner. Furthermore, by comparing the quantification of phosphorylation in cells exposed to various kinase inhibitors, the authors assigned specific kinases to many of phosphorylation events, which identified points of crosstalk between signaling pathways. This study provides proof of principle that this approach enables simultaneous analysis of phosphorylation events in multiple samples from different time points, doses, or patients. A major goal in signaling biology is the establishment of high-throughput quantitative methods for measuring changes in protein phosphorylation of entire signal transduction pathways across many different samples comprising temporal or dose data or patient samples. Data-independent acquisition (DIA) mass spectrometry (MS) methods, which involve tandem MS scans that are collected independently of precursor ion information and then are followed by targeted searching for known peptides, may achieve this goal. We applied DIA-MS to systematically quantify phosphorylation of components in the insulin signaling network in response to insulin as well as in stimulated cells exposed to a panel of kinase inhibitors targeting key downstream effectors in the network. We accurately quantified the effect of insulin on phosphorylation of 86 protein targets in the insulin signaling network using either stable isotope standards (SIS) or label-free quantification (LFQ) and mapped signal transmission through this network. By matching kinases to specific phosphorylation events (based on linear consensus motifs and temporal phosphorylation) to the quantitative phosphoproteomic data from cells exposed to inhibitors, we investigated predicted kinase-substrate relationships of AKT and mTOR in a targeted fashion. Furthermore, we applied this approach to show that AKT2-dependent phosphorylation of GAB2 promoted insulin signaling but inhibited epidermal growth factor (EGF) signaling in a manner dependent on 14-3-3 binding. Because DIA-MS can increase throughput and improve the reproducibility of peptide detection across multiple samples, this approach should facilitate more accurate, comprehensive, and quantitative assessment of signaling networks under various experimental conditions than are possible using other MS proteomic methods.