Targeted proteome investigation via selected reaction monitoring mass spectrometry

Due to the enormous complexity of proteomes which constitute the entirety of protein species expressed by a certain cell or tissue, proteome-wide studies performed in discovery mode are still limited in their ability to reproducibly identify and quantify all proteins present in complex biological samples. Therefore, the targeted analysis of informative subsets of the proteome has been beneficial to generate reproducible data sets across multiple samples. Here we review the repertoire of antibody- and mass spectrometry (MS) -based analytical tools which is currently available for the directed analysis of predefined sets of proteins. The topics of emphasis for this review are Selected Reaction Monitoring (SRM) mass spectrometry, emerging tools to control error rates in targeted proteomic experiments, and some representative examples of applications. The ability to cost- and time-efficiently generate specific and quantitative assays for large numbers of proteins and posttranslational modifications has the potential to greatly expand the range of targeted proteomic coverage in biological studies. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry

The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.     

Range of protein detection by selected/multiple reaction monitoring mass spectrometry in an unfractionated human cell culture lysate

Selected or multiple reaction monitoring is a targeted mass spectrometry method (S/MRM-MS), in which many peptides are simultaneously and consistently analyzed during a single liquid chromatography-mass spectrometry (LC-S/MRM-MS) measurement. These capabilities make S/MRM-MS an attractive method to monitor a consistent set of proteins over various experimental conditions. To increase throughput for S/MRM-MS it is advantageous to use scheduled methods and unfractionated protein extracts. Here, we established the practically measurable dynamic range of proteins reliably detectable and quantifiable in an unfractionated protein extract from a human cell line using LC-S/MRM-MS. Initially, we analyzed S/MRM transition peak groups in terms of interfering signals and compared S/MRM transition peak groups to MS1-triggered MS2 spectra using dot-product analysis. Finally, using unfractionated protein extract from human cell lysate, we quantified the upper boundary of copies per cell to be 35 million copies per cell, while 7500 copies per cell represents a lower boundary using a single 35 min linear gradient LC-S/MRM-MS measurement on a current, standard commercial instrument.     

Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry

Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications.     

Targeted proteomics by selected reaction monitoring mass spectrometry: applications to systems biology and biomarker discovery

Mass Spectrometry-based proteomics is now considered a relatively established strategy for protein analysis, ranging from global expression profiling to the identification of protein complexes and specific post-translational modifications. Recently, Selected Reaction Monitoring Mass Spectrometry (SRM-MS) has become increasingly popular in proteome research for the targeted quantification of proteins and post-translational modifications. Using triple quadrupole instrumentation (QqQ), specific analyte molecules are targeted in a data-directed mode. Used routinely for the quantitative analysis of small molecular compounds for at least three decades, the technology is now experiencing broadened application in the proteomics community. In the current review, we will provide a detailed summary of current developments in targeted proteomics, including some of the recent applications to biological research and biomarker discovery.     

Evaluation of absolute peptide quantitation strategies using selected reaction monitoring

The use of internal peptide standards in selected reaction monitoring experiments enables absolute quantitation. Here, we describe three approaches addressing calibration of peptide concentrations in complex matrices and assess their performance in terms of trueness and precision. The simplest approach described is single reference point quantitation where a heavy peptide is spiked into test samples and the endogenous analyte quantified relative to the heavy peptide internal standard. We refer to the second approach as normal curve quantitation. Here, a constant amount of heavy peptide and a varying amount of light peptide are spiked into matrix to construct a calibration curve. This accounts for matrix effects but due to the presence of endogenous analyte, it is usually not possible to determine the lower LOQ. We refer to the third method as reverse curve quantitation. Here, a constant amount of light peptide and a varying amount of heavy peptide are spiked into matrix to construct a calibration curve. Because there is no contribution to the heavy peptide signal from endogenous analyte, it is possible to measure the equivalent of a blank sample and determine LOQ. These approaches are applied to human plasma samples and used to assay peptides of a set of apolipoproteins.     

Quantitative analysis by liquid chromatography-tandem mass spectrometry of deuterium-labeled and unlabeled vitamin E in biological samples

A method for quantification of unlabeled alpha-tocopherol and the deuterated tocopherols, RRR-alpha-5-(CD(3))-tocopherol (d(3)RRR) and all rac-alpha-5,7-(CD(3))(2) tocopherol (d(6)all-rac) in plasma by HPLC-tandem mass spectrometry (LC-MS/MS) has been developed. Deuterated and unlabeled alpha-tocopherols were separated by HPLC and were detected by positive ion multiple-reaction monitoring using a triple-quadrupole mass spectrometer equipped with a heated nebulizer-atmospheric pressure chemical ionization interface, following routine extraction of vitamin E from plasma. The accuracy and precision were evaluated by replicate analysis of standards and samples. Human plasma samples, which were obtained at different times after the subject had consumed a capsule containing 1:1 ratio of d(3)RRR and d(6)all rac-alpha-tocopheryl acetates, were analyzed with this method. Plasma deuterated alpha-tocopherols measured by LC-MS/MS followed the same pattern as previously demonstrated by GC-MS measurement, without requiring an extra derivitization step. The detection limit was 10 pmol for each form of alpha-tocopherol injected.     

Development of protein biomarkers in cerebrospinal fluid for secondary progressive multiple sclerosis using selected reaction monitoring mass spectrometry (SRM-MS)

ABSTRACT: BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). It involves damage to the myelin sheath surrounding axons and to the axons themselves. MS most often presents with a series of relapses and remissions but then evolves over a variable period of time into a slowly progressive form of neurological dysfunction termed secondary progressive MS (SPMS). The reasons for this change in clinical presentation are unclear. The absence of a diagnostic marker means that there is a lag time of several years before the diagnosis of SPMS can be established. At the same time, understanding the mechanisms that underlie SPMS is critical to the development of rational therapies for this untreatable stage of the disease. RESULTS: Using LC coupled mass spectrometry; we have established a highly specific and sensitive multiplex selected reaction monitoring (SRM) assay. Our SRM assay has facilitated the simultaneous detection of surrogate peptides originating from 28 proteins present in cerebrospinal fluid (CSF). Protein levels in CSF are generally ~200-fold lower than that in human sera. A limit of detection (LOD) was determined to be as low as one femtomole per uL. We processed and analysed CSF samples from a total of 22 patients with SPMS, 12 patients with non-inflammatory neurological disorders (NIND) and 10 age-matched healthy controls in parallel for the levels of 28 selected potential protein biomarkers, followed by principal component analysis (PCA) for clustering protein biomarkers. Our SRM data suggested different levels of agrin, kallikrein and putative myosin-XVB in SPMS patients as compared to healthy controls. PCA reveals that these proteins are correlated, can be grouped into four principal components. Overall, we established an efficient platform to verify protein biomarkers in CSF, which can be easily adapted to other proteins of interest related to neurodegenerative diseases. CONCLUSIONS: A highly specific and sensitive multiplex SRM-MS assay was established for verifying CSF protein biomarkers in SPMS. Three proteins were found to be expressed significantly differently in SPMS patients as compared to health controls, which will help further our current understanding of SPMS disease pathology and/or therapeutic intervention.     

Simultaneous determination of ketobemidone and its N-demethylated metabolite in patient plasma samples by gas chromatography mass spectrometry with selected ion monitoring

An analytical procedure has been developed for the simultaneous determination of ketobemidone and its N-demethylated metabolite, norketobemidone. After isolation from plasma and re-extraction to acidic aqueous phase, the two aminophenols were extracted as ions pairs with tetrabutylammonium to dichloromethane, where derivatization with ethyl chloroformate took place. Determination was performed by gas chromatography mass spectrometry with selected ion monitoring. Ketobemidone and norketobemidone could be detected in plasma in a concentration of 1 ng ml-1 and 3 ng ml-1, respectively. Determinations were performed down to 5 ng ml-1. The relative standard deviation of the method in the analysis of 10 ng ml-1 of ketobemidone and norketobemidone, respectively, was 8% and 9% (n=10). The absolute recovery of unconjugated ketobemidone and norketobemidone through the method at the 100 ng ml-1 level was 91% and 85%, respectively. The method was applied to the determination of ketobemidone and norketobemidone in plasma from patients given ketobemidone. The concentrations of unconjugated norketobemidone was too small to be detected.     

Combining selected reaction monitoring with discovery proteomics in limited biological samples

Simultaneous quantification of multiple proteins by selected reaction monitoring (SRM) has several applications in cell signaling studies including embryo proteomics. However, concerns have recently been raised over the specificity of SRM assays due to possible ion redundancy and/or sequence similarity of selected peptide with multiple non‐related proteins. In this Viewpoint article, we discuss some simple measures that can increase our confidence in the accuracy of SRM scans used in proteomic experiments. At least in embryonic samples from porcine species, these measures were found to be useful in validating MS‐identified differentially expressed proteins. Among the nine proteins analyzed by SRM assay, all the proteins that were found to be up‐ or down‐regulated in MS experiment were also faithfully up‐ or down‐regulated in SRM assay.     

Enantiomeric composition analysis of pranoprofen in equine plasma and urine by chiral liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode

The enantioseparation of pranoprofen after its addition in racemic form into equine plasma and urine was conducted by chiral liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. The methods for the assay of both enantiomers were linear (r≥0.9943) in the low range from 0.001 to 0.1μg/mL and high range from 0.01 to 1.0μg/mL with good precision (% RSD≤5.6) and accuracy (% RE=-5.3 to 1.9). When racemic pranoprofen was orally administered to four horses at a single dose of 3.1mg/kg, the median plasma concentrations of (R)-pranoprofen were lower than the levels of (S)-pranoprofen from start to finish. In contrast, the urinary level of (R)-pranoprofen was 2.5 fold higher than (S)-pranoprofen level for the first 6h, followed by its rapid decrease down below (S)-pranoprofen concentration. Monitoring of the R/S ratios in equine urine may be useful for the prevention of false positive in horse doping test.     

Investigations of animal blood samples after fragrance drug inhalation by gas chromatography/mass spectrometry with chemical ionization and selected ion monitoring.

The fragrance compounds linalool (1) and linalyl acetate (2) could be detected, identified and quantified (1: 7-9 ng ml-1; and 2: 1-2 ng ml-1 and 4-5 ng ml-1 as free linalool) in blood samples after inhalation in animal experiments (mice) by gas chromatography/mass spectrometry (GC/MS) with chemical ionization (CI) (ammonia); selected ion monitoring (SIM) mode (1: m/z 81, 137 and 154; 2: 47, 57 and 137) and GC/flame ionization detection (FID). The inhalation of these monoterpenes in concentrations of 5 mg l-1 air leads to a significant reduction of the motility of the test animals down to 30-40% with respect to the control group.     

Automated selected reaction monitoring software for accurate label-free protein quantification

Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.     

ATAQS: A computational software tool for high throughput transition optimization and validation for selected reaction monitoring mass spectrometry

Background

Copy number variants (CNVs), including deletions, amplifications, and other rearrangements, are common in human and cancer genomes. Copy number data from array comparative genome hybridization (aCGH) and next-generation DNA sequencing is widely used to measure copy number variants. Comparison of copy number data from multiple individuals reveals recurrent variants. Typically, the interior of a recurrent CNV is examined for genes or other loci associated with a phenotype. However, in some cases, such as gene truncations and fusion genes, the target of variant lies at the boundary of the variant.

Results

We introduce Neighborhood Breakpoint Conservation (NBC), an algorithm for identifying rearrangement breakpoints that are highly conserved at the same locus in multiple individuals. NBC detects recurrent breakpoints at varying levels of resolution, including breakpoints whose location is exactly conserved and breakpoints whose location varies within a gene. NBC also identifies pairs of recurrent breakpoints such as those that result from fusion genes. We apply NBC to aCGH data from 36 primary prostate tumors and identify 12 novel rearrangements, one of which is the well-known TMPRSS2-ERG fusion gene. We also apply NBC to 227 glioblastoma tumors and predict 93 novel rearrangements which we further classify as gene truncations, germline structural variants, and fusion genes. A number of these variants involve the protein phosphatase PTPN12 suggesting that deregulation of PTPN12, via a variety of rearrangements, is common in glioblastoma.

Conclusions

We demonstrate that NBC is useful for detection of recurrent breakpoints resulting from copy number variants or other structural variants, and in particular identifies recurrent breakpoints that result in gene truncations or fusion genes. Software is available at http://http.//cs.brown.edu/people/braphael/software.html.     

Platform for establishing interlaboratory reproducibility of selected reaction monitoring-based mass spectrometry peptide assays

Mass spectrometry (MS) is an attractive alternative to quantification of proteins by immunoassays, particularly for protein biomarkers of clinical relevance. Reliable quantification requires that the MS-based assays are robust, selective, and reproducible. Thus, the development of standardized protocols is essential to introduce MS into clinical research laboratories. The aim of this study was to establish a complete workflow for assessing the transferability and reproducibility of selected reaction monitoring (SRM) assays between clinical research laboratories. Four independent laboratories in North America, using identical triple-quadrupole mass spectrometers (Quantum Ultra, Thermo), were provided with standard protocols and instrumentation settings to analyze unknown samples and internal standards in a digested plasma matrix to quantify 51 peptides from 39 human proteins using a multiplexed SRM assay. The interlaboratory coefficient of variation (CV) was less than 10% for 25 of 39 peptides quantified (12 peptides were not quantified based upon hydrophobicity) and exhibited CVs less than 20% for the remaining peptides. In this report, we demonstrate that previously developed research platforms for SRM assays can be improved and optimized for deployment in clinical research environments.     

The selected reaction monitoring/multiple reaction monitoring-based mass spectrometry approach for the accurate quantitation of proteins: clinical applications in the cardiovascular diseases

Selected reaction monitoring, also known as multiple reaction monitoring, is a powerful targeted mass spectrometry approach for a confident quantitation of proteins/peptides in complex biological samples. In recent years, its optimization and application have become pivotal and of great interest in clinical research to derive useful outcomes for patient care. Thus, selected reaction monitoring/multiple reaction monitoring is now used as a highly sensitive and selective method for the evaluation of protein abundances and biomarker verification with potential applications in medical screening. This review describes technical aspects for the development of a robust multiplex assay and discussing its recent applications in cardiovascular proteomics: verification of promising disease candidates to select only the highest quality peptides/proteins for a preclinical validation, as well as quantitation of protein isoforms and post-translational modifications.