Multiple Reaction Monitoring Experiments Research Articles

Quantitative Mass Spectrometry by Isotope Dilution and Multiple Reaction Monitoring (MRM).

Selected reaction monitoring (SRM) is used in molecular profiling to detect and quantify specific known proteins in complex mixtures. Using isotope dilution (Barnidge et al., Anal Chem 75(3):445-451, 2003) methodologies, peptides can be quantified without the need for an antibody-based method. Selected reaction monitoring assays employ electrospray ionization mass spectrometry (ESI-MS) followed by two stages of mass selection: a first stage where the mass of the peptide ion is selected and, after fragmentation by collision-induced dissociation (CID), a second stage (tandem MS) where either a single (e.g., SRM) or multiple (multiple reaction monitoring, MRM) specific peptide fragment ions are transmitted for detection. The MRM experiment is accomplished by specifying the parent masses of the selected endogenous and isotope-labeled peptides for MS/MS fragmentation and then monitoring fragment ions of interest, using their intensities/abundances and relative ratios to quantify the parent protein of interest. In this example protocol, we will utilize isotope dilution MRM-MS to quantify in absolute terms the total levels of the protein of interest, ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Ataxia telangiectasia mutated (ATM) phosphorylates several key proteins that initiate activation of the DNA damage checkpoint leading to cell cycle arrest.

Quali-quantitative analysis of the phenolic fraction of the flowers of Corylus avellana, source of the Italian PGI product “Nocciola di Giffoni”: Isolation of antioxidant diarylheptanoids

There is only limited information available on the chemical composition of the non-edible parts of Corylus avellana, source of the Italian PGI product “Nocciola di Giffoni” (hazelnut). An initial LC-MS profile of the methanolic extract of the male flowers of C. avellana, cultivar ‘Tonda di Giffoni’ led to the isolation of 12 compounds, of which the structures were elucidated by NMR spectroscopy. These were identified as three previously undescribed diarylheptanoids, named giffonins Q-S, along with nine known compounds. Furthermore, the quantitative determination of the main compounds occurring in the methanolic extract of C. avellana flowers was carried out by an analytical approach based on LC-ESI(QqQ)MS, using the Multiple Reaction Monitoring (MRM) experiment. In order to explore the antioxidant ability of C. avellana flowers, the methanolic extract and the isolated compounds were evaluated for their inhibitory effects on human plasma lipid peroxidation induced by H2O2 and H2O2/Fe2+, by measuring the concentration of TBARS.

Comprehensive two-dimensional liquid chromatography–tandem mass spectrometry for the simultaneous determination of wine polyphenols and target contaminants

A novel system for comprehensive two-dimensional liquid chromatography coupled to a triple quadrupole mass spectrometer is described for the analysis of wine components. The first dimension consisted of a 250-mm microbore cyano column utilizing 5μm diameter particles, interfaced to a 50-mm superficially-porous particle C18 column with 2.7μm diameter particles. Both columns were operated under reversed-phase conditions. Correlation between the two chromatographic separation modes was decreased by designing a 60-s shift gradient program in the second dimension, and the increase in orthogonality was evaluated quantitatively utilizing a number of orthogonality metrics. The system was employed for the analysis of a red wine sample, without preliminary clean-up procedures, and a total of 43 polyphenols were separated and identified. Comparison with a one-dimensional LC system showed a large increase in the number of identified components with the two-dimensional system. Optimized multiple reaction monitoring experiments allowed for the determination of trans-resveratrol, which is one of the most active antioxidant component of wine, and for monuron, a plant protection product (herbicide) of interest to regulatory agencies. The estimated limits of detection and of quantification were 0.3μgL−1 and 1μgL−1, respectively, well below the minimum detection limit (10μgL−1) set by current regulation.

Erratum for: Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth

A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02 % trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2 % to 20 %, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth

A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02% trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2% to 20%, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

An extensive library of surrogate peptides for all human proteins

Selecting the most appropriate surrogate peptides to represent a target protein is a major component of experimental design in Multiple Reaction Monitoring (MRM). Our software PeptidePicker with its v-score remains distinctive in its approach of integrating information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM that is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our “best knowledge” for selecting candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it has previously been observed. Here we present an updated approach where we have already compiled a list of all possible surrogate peptides of the human proteome. Using our stringent selection criteria, the list includes 165k suitable MRM peptides covering 17k proteins of the human reviewed proteins in UniProtKB. Compared to average of 2–4min per protein for retrieving and integrating the information, the precompiled list includes all peptides available instantly. This allows a more cohesive and faster design of a multiplexed MRM experiment and provides insights into evidence for a protein's existence. We will keep this list up-to-date as proteomics data repositories continue to grow.This article is part of a Special Issue entitled: Computational Proteomics.

Application of high-resolution mass spectrometry to measure low abundance isotope enrichment in individual muscle proteins.

Stable isotope-labeled amino acids have long been used to measure the fractional synthesis rate of proteins, although the mass spectrometry platforms used for such analyses have changed throughout the years. More recently, tandem mass spectrometers such as triple quadrupoles have been accepted as the standard platform for enrichment measurement due to their sensitivity and the enhanced specificity offered by multiple reaction monitoring (MRM) experiments. The limit in the utility of such platforms for enrichment analysis occurs when measuring very low levels of enrichment from small amounts of sample, particularly proteins isolated from two-dimensional gel electrophoresis (2D-GE), where interference from contaminant ions impacts the sensitivity of the measurement. We therefore applied a high-resolution orbitrap mass spectrometer to the analysis of [ring-(13)C6]-phenylalanine enrichment in individual muscle proteins isolated with 2D-GE. Comparison of samples analyzed on both platforms revealed that the high-resolution MS has significantly improved sensitivity relative to the triple quadrupole MS at very low-level enrichments due to its ability to resolve interferences in the m/z dimension. At higher enrichment levels, enrichment measurements from the orbitrap platform showed significant correlation (R (2) > 0.5) with those of the triple quadrupole platform. Together, these results indicate that high-resolution MS platforms such as the orbitrap are not only as capable of performing isotope enrichment measurements as the more commonly preferred triple quadrupole instruments, but offer unparalleled advantages in terms of mass accuracy and sensitivity in the presence of similar-mass contaminants.

Discriminating precursors of common fragments for large-scale metabolite profiling by triple quadrupole mass spectrometry.

The goal of large-scale metabolite profiling is to compare the relative concentrations of as many metabolites extracted from biological samples as possible. This is typically accomplished by measuring the abundances of thousands of ions with high-resolution and high mass accuracy mass spectrometers. Although the data from these instruments provide a comprehensive fingerprint of each sample, identifying the structures of the thousands of detected ions is still challenging and time intensive. An alternative, less-comprehensive approach is to use triple quadrupole (QqQ) mass spectrometry to analyze predetermined sets of metabolites (typically fewer than several hundred). This is done using authentic standards to develop QqQ experiments that specifically detect only the targeted metabolites, with the advantage that the need for ion identification after profiling is eliminated. Here, we propose a framework to extend the application of QqQ mass spectrometers to large-scale metabolite profiling. We aim to provide a foundation for designing QqQ multiple reaction monitoring (MRM) experiments for each of the 82 696 metabolites in the METLIN metabolite database. First, we identify common fragmentation products from the experimental fragmentation data in METLIN. Then, we model the likelihoods of each precursor structure in METLIN producing each common fragmentation product. With these likelihood estimates, we select ensembles of common fragmentation products that minimize our uncertainty about metabolite identities. We demonstrate encouraging performance and, based on our results, we suggest how our method can be integrated with future work to develop large-scale MRM experiments. Our predictions, Supplementary results, and the code for estimating likelihoods and selecting ensembles of fragmentation reactions are made available on the lab website at http://pattilab.wustl.edu/FragPred.

146 QUANTIFICATION OF PEPTIDE GROWTH FACTORS IN CATTLE UTERINE FLUID BY MULTIPLE REACTION MONITORING-LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Multiple reaction monitoring (MRM) allows targeted quantitative proteomics with a wide dynamic range and limit of detection down to femtomoles. We used MRM to study uterine growth factors (GF) presumed to promote embryonic development. A validated experimental model was used to recover uterine fluid (UF) and analyse GF expression in the presence or absence of embryos. Briefly, Day-6 in vitro-produced embryos (n = 50) or vehicle (sham transfer) were transferred into the uteri of each oestrus-synchronized Holstein heifer (n = 14) during nonconsecutive cycles. Blood P4 concentrations were measured on Days 0 (oestrus), 6, and 8. On Day 8, UF was recovered from embryo and sham recipients. After retrieval, UF were centrifuged and supernatants stored at –145°C. Sham and embryo UF selected for MRM were from n = 10 animals (n = 20 samples). Uterine fluid, recovered after embryo transfer, contained on average n = 43.1 ± 5.2 total and n = 34.1 ± 3.7% viable embryos per recipient. For MRM, UF samples were concentrated, and protein was precipitated and resuspended in ammonium bicarbonate. Protein (20 μg) was reduced with DTT, trypsin-digested, and desalted. Proteotypic peptides for targeted GF were selected with MRM Pilot software (ABsciex, Farmingham, MA, USA), with 3 to 5 transitions programmed for each peptide. A control, unrelated synthetic peptide was spiked as an internal standard. The area of the larger transition for the control peptide was used to normalise the area values of each other peptide. The MRM experiments were performed on a 5500 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (ABsciex) equipped with an Eksigent 1D+plus nanoLC chromatographic system. Data analysis was performed with Analyst 1.5.2 and MultiQuant 2.0.2 softwares (ABsciex). The area of most abundant transition for each analysed peptide was used for relative quantitation. Proteins studied were betacellulin, heparin-binding EGF-like growth factor, neuregulin, artemin, connective tissue growth factor, nerve growth factor, kit ligand, stanniocalcin-1 (STC1), early pregnancy factor (EPF), and hepatoma-derived growth factor (HDGF). Proteotypic peptides were identified in all samples for HDGF, kit ligand, STC1, and EPF (n = 1, n = 1, n = 1, and n = 3 peptides, respectively), which precluded the analysis of the remaining GF. No differences in relative abundance were detected between UF containing or not containing embryos for HDGF, kit ligand, STC1, and EPF (2.85 ± 0.6 v. 4.43 ± 0.6; 0.15 ± 0.02 v. 0.16 ± 0.02; 0.03 ± 0.00 v. 0.04 ± 0.00; and 1.20 ± 0.16 v. 1.09 ± 1.16, respectively). However, STC1 and Day 8 blood P4 were highly correlated (r = 0.71; P = 0.0004), suggesting P4 regulation of STC1. Multiple reaction monitoring-LC-MS/MS is a useful technique to identify some scarce GF in UF at different dynamic ranges. MICINN, project AGL2012-37772 and FEDER. E. C. was supported by MEC-FPU-AP2009-5265. The authors are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception environment.

In-time and in-space tandem mass spectrometry to determine the metabolic profiling of flavonoids in a typical sweet cherry (Prunus avium L.) cultivar from Southern Italy.

This paper presents a comprehensive analytical methodology, based on 'in-time' and 'in-space' tandem mass spectrometry (MS) techniques, to identify and quantify flavonoid compounds in a typical Italian sweet cherry cultivar (cv. Ferrovia). Five anthocyanins, four flavan-3-ols and nine flavonols were determined by means of hyphenated high-performance liquid chromatography - multi-stage MS (HPLC-MS(n)) analyses (MS(n) up to MS(4)), among which quercetin-3-O-rutinoside-7-O-glucoside, kaempferol-3-O-rutinoside-7-O-glucoside, quercetin-3-O-galactosyl-rhamnoside and quercetin-3-O-coumaroylglucoside were tentatively identified in sweet cherries for the first time. Ultrafast HPLC and tandem MS (UHPLC-MS/MS) analyses through multiple reaction monitoring experiments showed that cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside were the main anthocyanins of cv. Ferrovia at maturity. Moreover, consistent levels of catechin and epicatechin as well as quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside were also found. Because flavonoids have been ascribed as potential health-promoting compounds, gathered findings provide new insight into the knowledge of the quali-quantitative profile of these phytochemicals into a widespread fruit such as sweet cherry.

A modified liquid chromatography/tandem mass spectrometry method for predominant disaccharide units of urinary glycosaminoglycans in patients with mucopolysaccharidoses.

BackgroundThe identification of acid mucopolysaccharide by the liquid chromatography/tandem mass spectrometry method (LC-MS/MS) of the predominant disaccharide units of glycosaminoglycans (GAGs) (chondroitin sulfate, CS; dermatan sulfate, DS; heparan sulfate, HS) after methanolysis is validated and applicable for mucopolysaccharidosis (MPS) type determination.MethodsA total of 76 urine samples were collected and analyzed, from nine MPS I patients, 13 MPS II patients, seven MPS III patients, eight MPS VI patients, and 39 normal controls. Urinary GAG was first precipitated by the Alcian blue method followed by a treatment of 3 N HCl methanol. The protonated species of the methylated disaccharide products were detected by using a multiple reaction monitoring experiment. Internal standards, [2H6] CS, [2H6] DS and [2H6] HS, were prepared in-house by deuteriomethanolysis of CS, DS and HS.ResultsOne particular disaccharide for each GAG was selected, in which the parent ion and its daughter ion after collision were m/z 426.1 → 236.2 for DS (m/z 432 → 239 for dimers derived from [2H6] CS and [2H6] DS) and m/z 384.2 → 161.9 for HS (m/z 390.4 → 162.5 for the [2H6] HS dimer). The quantities of DS and HS were determined, which varied from one MPS type to the other. The results can be used to evaluate the severity of MPS subgroups, as well as urinary GAG amelioration at follow-up after enzyme replacement therapy (ERT).ConclusionsThe modified LC–MS/MS method for MPS type determination is specific, sensitive, validated, accurate, and applicable for simultaneous quantifications of urinary DS and HS. This method can help to make correct diagnosis of MPS patients and evaluate the effectiveness of ERT.

New Sensitive High-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for the Detection of Horse and Pork in Halal Beef

The accidental or fraudulent blending of meat from different species is a highly relevant aspect for food product quality control, especially for consumers with ethical concerns against species, such as horse or pork. In this study, we present a sensitive mass spectrometrical approach for the detection of trace contaminations of horse meat and pork and demonstrate the specificity of the identified biomarker peptides against chicken, lamb, and beef. Biomarker peptides were identified by a shotgun proteomic approach using tryptic digests of protein extracts and were verified by the analysis of 21 different meat samples from the 5 species included in this study. For the most sensitive peptides, a multiple reaction monitoring (MRM) method was developed that allows for the detection of 0.55% horse or pork in a beef matrix. To enhance sensitivity, we applied MRM(3) experiments and were able to detect down to 0.13% pork contamination in beef. To the best of our knowledge, we present here the first rapid and sensitive mass spectrometrical method for the detection of horse and pork by use of MRM and MRM(3).

Plasma lipid profiling in a large population-based cohort

We have performed plasma lipid profiling using liquid chromatography electrospray ionization tandem mass spectrometry on a population cohort of more than 1,000 individuals. From 10 μl of plasma we were able to acquire comparative measures of 312 lipids across 23 lipid classes and subclasses including sphingolipids, phospholipids, glycerolipids, and cholesterol esters (CEs) in 20 min. Using linear and logistic regression, we identified statistically significant associations of lipid classes, subclasses, and individual lipid species with anthropometric and physiological measures. In addition to the expected associations of CEs and triacylglycerol with age, sex, and body mass index (BMI), ceramide was significantly higher in males and was independently associated with age and BMI. Associations were also observed for sphingomyelin with age but this lipid subclass was lower in males. Lysophospholipids were associated with age and higher in males, but showed a strong negative association with BMI. Many of these lipids have previously been associated with chronic diseases including cardiovascular disease and may mediate the interactions of age, sex, and obesity with disease risk.

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. 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.

Bioassay-Guided Isolation of a Low Molecular Weight PHB from Burkholderia sp. with Phytotoxic Activity

This work reports on the bioassay-guided isolation and identification of the macrocyclic pentolide 1, a cyclic polyhydroxybutyrate (PHB) with low molecular weight. This metabolite is produced by Burkholderia sp. and it exhibited phytotoxic activity in a Lemna minor bioassay. Its structure was determined by (1)H and (13)C NMR, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, IR, and electrospray ionization tandem mass spectrometry analyses. The period for maximum production of the pentolide was optimized and determined on the basis of multiple reaction monitoring experiments at 15 days. The potential of Burkholderia sp. as a producer of higher biopolymers of PHB was also investigated. The methodology employed here accelerated the isolation and characterization of a phytotoxic metabolite whose structure can serve as a model for the synthesis of new classes of herbicides.

Direct analysis of 4-methylimidazole in foods using paper spray mass spectrometry

Rapid qualitative and quantitative analysis of 4-methylimidazole (4-MEI) in caramel and beverage samples is demonstrated using the paper spray form of ambient ionization mass spectrometry. The minimum level of pure 4-MEI detectable using multiple reaction monitoring (MRM) in a triple quadrupole instrument was 3 pg μL⁻¹ in neat solvent and 5 pg μL⁻¹ in a matrix containing caramel. This method was used to analyze 11 caramel samples for 4-MEI. After implementing effective quality control protocols, average relative standard deviations for paper spray triple quadrupole MS were less than 15% and the linear dynamic range was three orders of magnitude. Results obtained on three different days by two different analyst groups agreed closely. An ion trap tandem MS method of approximate quantitative analysis is also described and it gave similar precision to the triple quadrupole experiment when fluctuations in ion currents were cancelled by simultaneously isolating the analyte and internal standard and fragmenting both in an ion trap simultaneous MRM experiment. As another alternative, a rapid qualitative screening method based on the use of high-resolution measurements instead of tandem mass spectrometry using an Orbitrap was also tested and found to give a detection limit of 100 pg μL⁻¹.

Quantification of ginsenosides Rh4 and Rk3 in rat plasma by liquid chromatography‐tandem mass spectrometry: Application to a pre‐clinical pharmacokinetic study

Ginsenoside Rh4 (Rh4) and ginsenoside Rk3 (Rk3) are two active substances isolated from the processed Panax species. To further explore their potential medicinal application, a reliable liquid chromatography-tandem mass spectrometry method (LC/MS/MS) was developed and validated for the quantification of Rh4 and Rk3 in rat plasma. Multiple ion monitoring and multiple reaction monitoring experiments were performed in negative ionization mode. This LC/MS/MS method had good selectivity, sensitivity (lower limit of quantification = 10 ng/mL), precision (intra- and inter-day relative standard deviation ≤ 10.1) and accuracy (analytical recovery within 100 ± 10%). The pharmacokinetic profiles of Rh4 and Rk3 were subsequently assessed in Sprague-Dawley rats. Similar to many other ginsenosides, the oral bioavailability of Rh4 and Rk3 was unfavorable, and Rh4 and Rk3 did not have any measurable plasma exposure after oral administration (20 mg/kg). Fortunately, upon intravenous administration (5 mg/kg), both Rh4 and Rk3 possessed abundant plasma exposure, moderate clearance (Cl = 50.2 ± 7.7 and 23.8 ± 1.4 mL·min(-1)·kg(-1), respectively) and terminal elimination half-life (t(1/2 λZ) = 157.2 ± 65.2 and 99.5 ± 37.8 min, respectively). As Rh4 and Rk3 displayed favorable intravenous pharmacokinetic profiles, further exploration on their medicinal application is warranted.

Targeted Proteomics for Determining Phosphorylation Site-Specific Associations in Cardiovascular Disease

Post-translational modifications (PTMs) are chemical or physical changes to proteins that can alter structure/function relationships and thus influence catalysis, cell localization and interactions with other proteins and biomolecules. There are over 200 recognized PTMs, generating a vast scope for altering protein function and increasing the potential for complex regulatory cross-talk between diverse modifications. Protein phosphorylation is of major significance since 30-50% of all cellular proteins may be targeted1,2. Phosphorylation is a transient means of amplifying environmental signals to rapidly alter protein structure, interactions and ultimately, function.

Quantification of glycopeptides by multiple reaction monitoring liquid chromatography/tandem mass spectrometry

Protein glycosylation has a major influence on functions of proteins. Studies have shown that aberrations in glycosylation are indicative of disease conditions. This has prompted major research activities for comparative studies of glycoproteins in biological samples. Multiple reaction monitoring (MRM) is a highly sensitive technique which has been recently explored for quantitative proteomics. In this work, MRM was adopted for quantification of glycopeptides derived from both model glycoproteins and depleted human blood serum using glycan oxonium ions as transitions. The utilization of oxonium ions aids in identifying the different types of glycans bound to peptide backbones. MRM experiments were optimized by evaluating different parameters that have a major influence on quantification of glycopeptides, which include MRM time segments, number of transitions, and normalized collision energies. The results indicate that oxonium ions could be adopted for the characterization and quantification of glycopeptides in general, eliminating the need to select specific transitions for individual precursor ions. Also, the specificity increased with the number of transitions and a more sensitive analysis can be obtained by providing specific time segments. This approach can be applied to comparative and quantitative studies of glycopeptides in biological samples as illustrated for the case of depleted blood serum sample. Copyright © 2012 John Wiley & Sons, Ltd.

Using iRT, a normalized retention time for more targeted measurement of peptides.

Multiple reaction monitoring (MRM) has recently become the method of choice for targeted quantitative measurement of proteins using mass spectrometry. The method, however, is limited in the number of peptides that can be measured in one run. This number can be markedly increased by scheduling the acquisition if the accurate retention time (RT) of each peptide is known. Here we present iRT, an empirically derived dimensionless peptide-specific value that allows for highly accurate RT prediction. The iRT of a peptide is a fixed number relative to a standard set of reference iRT-peptides that can be transferred across laboratories and chromatographic systems. We show that iRT facilitates the setup of multiplexed experiments with acquisition windows more than four times smaller compared to in silico RT predictions resulting in improved quantification accuracy. iRTs can be determined by any laboratory and shared transparently. The iRT concept has been implemented in Skyline, the most widely used software for MRM experiments.