Improved precision of iTRAQ and TMT quantification by an axial extraction field in an Orbitrap HCD cell.
ABSTRACT: Improving analytical precision is a major goal in quantitative differential proteomics as high precision ensures low numbers of outliers, a source of false positives with regard to quantification. In addition, higher precision increases statistical power, i.e., the probability to detect significant differences. With chemical labeling using isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tag (TMT) reagents, quantification is based on the extraction of reporter ions from tandem mass spectrometry (MS/MS) spectra. We compared the performance of two versions of the LTQ Orbitrap higher energy collisional dissociation (HCD) cell with and without an axial electric field with regard to reporter ion quantification. The HCD cell with the axial electric field was designed to push fragment ions into the C-trap and this version is mounted in current Orbitrap XL ETD and Orbitrap Velos instruments. Our goal was to evaluate whether the purported improvement in ion transmission had a measurable impact on the precision of MS/MS based quantification using peptide labeling with isobaric tags. We show that the axial electric field led to an increased percentage of HCD spectra in which the complete set of reporter ions was detected and, even more important, to a reduction in overall variance, i.e., improved analytical precision of the acquired data. Notably, adequate precision of HCD-based quantification was maintained even for low precursor ion intensities of a complex biological sample. These findings may help researchers in their design of quantitative proteomics studies using isobaric tags and establish HCD-based quantification on the LTQ Orbitrap as a highly precise approach in quantitative proteomics.
Project description:Isobaric tags enable multiplexed quantitative analysis of many biological samples in a single LC-MS/MS experiment. As a cost-effective alternative to expensive commercial isobaric tagging reagents, we developed our own custom N,N-dimethylleucine "DiLeu" isobaric tags for quantitative proteomics. Here, we present a new generation of DiLeu tags that achieves 21-plex quantification in high-resolution HCD MS/MS spectra via distinct reporter ions that differ in mass from each other by a minimum of 3 mDa. The 21-plex set retains the compact tag structure and existing isotopologues of the 12-plex set but includes nine new reporter variants formulated with unique configurations of 13C, 15N, and 2H stable isotopes, each synthesized in-house via a stepwise N-monomethylation synthesis strategy using readily available reagents. Thus, multiplexing capacity is expanded significantly, while preserving the performance and low cost of the previous implementation. We show that 21-plex DiLeu tags generate strong reporter ions following HCD fragmentation of labeled peptides acquired on Orbitrap platforms at a minimum of 60,000 resolving power (at 400 m/z), and we demonstrate accurate 21-plex quantification of labeled K562 human cell line protein digests via single-shot nanoLC-MS/MS analysis on a Q Exactive HF system.
Project description:Isobaric stable isotope tagging reagents such as tandem mass tags or isobaric tags for relative and absolute quantification enable multiplexed quantification of peptides via reporter ion signals in the low mass range of tandem mass spectra. Until recently, the poor recovery of low mass fragments observed in tandem mass spectra acquired on ion trap mass spectrometers precluded the use of these reagents on this widely available instrument platform. The Pulsed Q Dissociation (PQD) technique allows negotiating this limitation but suffers from poor fragmentation efficiency, which has raised doubts in the community as to its practical utility. Here we show that by carefully optimizing instrument parameters such as collision energy, activation Q, delay time, ion isolation width, number of microscans, and number of trapped ions, low m/z fragment ion intensities can be generated that enable accurate peptide quantification at the 100 amol level. Side by side comparison of PQD on an LTQ Orbitrap with CID on a five-year old Q-Tof Ultima using complex protein digests shows that whereas precision of quantification of 10-15% can be achieved by both approaches, PQD quantifies twice as many proteins. PQD on an LTQ Orbitrap also outperforms "higher energy collision induced dissociation" on the same instrument using the recently introduced octapole collision cell in terms of lower limit of quantification. Finally, we demonstrate the significant analytical potential of iTRAQ quantification using PQD on an LTQ Orbitrap by quantitatively measuring the kinase interaction profile of the small molecule drug imatinib in K-562 cells. This article gives practical guidance for the implementation of PQD, discusses its merits, and for the first time, compares its performance to higher energy collision-induced dissociation.
Project description:The recently developed and commercially available carbonyl-reactive tandem mass tags (aminoxyTMT) enable multiplexed quantification of glycans through comparison of reporter ion intensities. However, challenges still exist for collision activated dissociation (CAD) MS/MS based quantification of aminoxyTMT due to the relatively low reporter ion yield especially for glycans with labile structures. To circumvent this limitation, we utilized the unique structural features of N-glycan molecules, the common core sugar sequence (HexNAc)2(Man)3, and common m/z of Yn ions generated from different types of precursors by MS/MS and designed a Y1 ion triggered, targeted MultiNotch MS3 relative quantification approach based on aminoxyTMT labeling. This approach was implemented on a nanoHILIC-Tribrid quadrupole-ion trap-Orbitrap platform, which enables prescreening of aminoxyTMT labeled N-glycan precursor ions by Y1 ion fragment ion mass in a higher-energy collisional dissociation (HCD) MS/MS scan and coisolation and cofragmentation of multiple Yn fragment ions that carry the isobaric tags from the inclusion list in the MS/MS/MS scan. Through systematical optimization and evaluation using N-glycans released from several glycoprotein standards and human serum proteins, we demonstrated that the Y1 ion triggered, targeted MultiNotch MS3 approach offers improved accuracy, precision, and sensitivity for relative quantification compared to traditional data-dependent MS2 and Y1 ion MS3 quantification methods.
Project description:Peptide labeling with isobaric tags has become a popular technique in quantitative shotgun proteomics. Using two different samples viz. a protein mixture and HeLa extracts, we show that three commercially available isobaric tags differ with regard to peptide identification rates: The number of identified proteins and peptides was largest with iTRAQ 4-plex, followed by TMT 6-plex, and smallest with iTRAQ 8-plex. In all experiments, we employed a previously described method where two scans were acquired for each precursor on an LTQ Orbitrap: A CID scan under standard settings for identification, and a HCD scan for quantification. The observed differences in identification rates were similar when data was searched with either Mascot or Sequest. We consider these findings to be the result of a combination of several factors, most notably prominent ions in CID spectra as a consequence of loss of fragments of the label tag from precursor ions. These fragment ions cannot be explained by current search engines and were observed to have a negative impact on peptide scores.
Project description:Multiplex isobaric tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ)) are a valuable tool for high-throughput mass spectrometry based quantitative proteomics. We have developed our own multiplex isobaric tags, DiLeu, that feature quantitative performance on par with commercial offerings but can be readily synthesized in-house as a cost-effective alternative. In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of (13)C, (15)N, and (2)H stable isotopes in the reporter group. The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation. We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography-tandem mass spectrometry (nanoLC-MS(2)) analysis on an Orbitrap Elite mass spectrometer.
Project description:While developing a multiplexed phosphotyrosine peptide quantification assay, an unexpected observation was made: significant neutral loss from phosphotyrosine (pY) containing peptides. Using a 2000-member peptide library, we sought to systematically investigate this observation by comparing unlabeled peptides with the two highest-plex isobaric tags (iTRAQ8 and TMT10) across CID, HCD, and ETD fragmentation using high resolution high mass accuracy Orbitrap instrumentation. We found pY peptide neutral loss behavior was consistent with reduced proton mobility, and does not occur during ETD. The site of protonation at the peptide N-terminus changes from a primary to a tertiary amine as a result of TMT labeling which would increase the gas phase basicity and reduce proton mobility at this site. This change in fragmentation behavior has implications during instrument method development and interpretation of MS/MS spectra, and therefore ensuing follow-up studies. We show how sites not localized to tyrosine by search and site localization algorithms can be confidently reassigned to tyrosine using neutral loss and phosphotyrosine immonium ions. We believe these findings will be of general interest to those studying pY signal transduction using isobaric tags.
Project description:Multiplex isobaric tags have become valuable tools for high-throughput quantitative analysis of complex biological samples in discovery-based proteomics studies. Hybrid labeling strategies that pair stable isotope mass difference labeling with multiplex isobaric tag-based quantification further facilitate these studies by greatly increasing multiplexing capability. In this work, we present a cost-effective chemical labeling approach that couples duplex stable isotope dimethyl labeling with our custom 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags in a combined precursor isotopic labeling and isobaric tagging (cPILOT) strategy that is compatible with a wide variety of biological samples and permits 24-plex quantification in a single LC-MS/MS experiment. We demonstrate the utility of the DiLeu cPILOT approach by labeling yeast digests and performing proof-of-principle quantification experiments on the Orbitrap Fusion Lumos.
Project description:Label-free peptide quantification in liquid chromatography-mass spectrometry (LC-MS) proteomics analyses is complicated by the presence of isobaric coeluting peptides, as they generate the same extracted ion chromatogram corresponding to the sum of their intensities. Histone proteins are especially prone to this, as they are heavily modified by post-translational modifications (PTMs). Their proteolytic digestion leads to a large number of peptides sharing the same mass, while carrying PTMs on different amino acid residues. We present an application of MS data-independent acquisition (DIA) to confidently determine and quantify modified histone peptides. By introducing the use of low-resolution MS/MS DIA, we demonstrate that the signals of 111 histone peptides could easily be extracted from LC-MS runs due to the relatively low sample complexity. By exploiting an LTQ-Orbitrap mass spectrometer, we parallelized MS and MS/MS scan events using the Orbitrap and the linear ion trap, respectively, decreasing the total scan time. This, in combination with large windows for MS/MS fragmentation (50 m/z) and multiple full scan events within a DIA duty cycle, led to a MS scan cycle speed of ?45 full MS per minute, improving the definition of extracted LC-MS chromatogram profiles. By using such acquisition method, we achieved highly comparable results to our optimized acquisition method for histone peptide analysis (R(2) correlation > 0.98), which combines data-dependent acquisition (DDA) and targeted MS/MS scans, the latter targeting isobaric peptides. By using DIA, we could also remine our data set and quantify 16 additional isobaric peptides commonly not targeted during DDA experiments. Finally, we demonstrated that by performing the full MS scan in the linear ion trap, we achieve highly comparable results as when adopting high-resolution MS scans (R(2) correlation 0.97). Taken together, results confirmed that histone peptide analysis can be performed using DIA and low-resolution MS with high accuracy and precision of peptide quantification. Moreover, DIA intrinsically enables data remining to later identify and quantify isobaric peptides unknown at the time of the LC-MS experiment. These methods will open up epigenetics analyses to the proteomics community who do not have routine access to the newer generation high-resolution MS/MS generating instruments.
Project description:Pulsed Q collision-induced dissociation (PQD) was developed in part to facilitate detection of low-mass reporter ions using labeling reagents (e.g. iTRAQ) on LTQ platforms. It has generally been recognized that the scan speed and sensitivity of an LTQ are superior than those of an Orbitrap using the higher-energy collisional dissociation (HCD). However, the use of PQD in quantitative proteomics is limited, primarily due to the meager reproducibility of reporter ion ratios. Optimizations of PQD for iTRAQ quantification using LTQ have been reported, but a universally applicable strategy for quantifying the less abundant proteins has not been fully established. Adjustments of the AGC target, ?scan, or scan speed offer only incremental improvements in reproducibility. From our experience, however, satisfactory coefficients of variation (CVs) of reporter ion ratios were difficult to achieve using the discovery-based approach. As an alternative, we implemented a target-based approach that obviates data dependency to allow repetitive data acquisitions across chromatographic peaks. Such a strategy generates enough data points for more reliable quantification. Using cAMP treatment in S49 cell lysates and this target-based approach, we were able to validate differentially expressed proteins, which were initially identified as potential candidates using the discovery-based PQD. The target-based strategy also yielded results comparable to those obtained from HCD in an Orbitrap. Our findings should aid LTQ users who desire to explore iTRAQ quantitative proteomics but have limited access to the more costly Orbitrap or other instruments.
Project description:The two key steps for analyzing proteomic data generated by high-resolution MS are database searching and postprocessing. While the two steps are interrelated, studies on their combinatory effects and the optimization of these procedures have not been adequately conducted. Here, we investigated the performance of three popular search engines (SEQUEST, Mascot, and MS Amanda) in conjunction with five filtering approaches, including respective score-based filtering, a group-based approach, local false discovery rate (LFDR), PeptideProphet, and Percolator. A total of eight data sets from various proteomes (e.g., E. coli, yeast, and human) produced by various instruments with high-accuracy survey scan (MS1) and high- or low-accuracy fragment ion scan (MS2) (LTQ-Orbitrap, Orbitrap-Velos, Orbitrap-Elite, Q-Exactive, Orbitrap-Fusion, and Q-TOF) were analyzed. It was found combinations involving Percolator achieved markedly more peptide and protein identifications at the same FDR level than the other 12 combinations for all data sets. Among these, combinations of SEQUEST-Percolator and MS Amanda-Percolator provided slightly better performances for data sets with low-accuracy MS2 (ion trap or IT) and high accuracy MS2 (Orbitrap or TOF), respectively, than did other methods. For approaches without Percolator, SEQUEST-group performs the best for data sets with MS2 produced by collision-induced dissociation (CID) and IT analysis; Mascot-LFDR gives more identifications for data sets generated by higher-energy collisional dissociation (HCD) and analyzed in Orbitrap (HCD-OT) and in Orbitrap Fusion (HCD-IT); MS Amanda-Group excels for the Q-TOF data set and the Orbitrap Velos HCD-OT data set. Therefore, if Percolator was not used, a specific combination should be applied for each type of data set. Moreover, a higher percentage of multiple-peptide proteins and lower variation of protein spectral counts were observed when analyzing technical replicates using Percolator-associated combinations; therefore, Percolator enhanced the reliability for both identification and quantification. The analyses were performed using the specific programs embedded in Proteome Discoverer, Scaffold, and an in-house algorithm (BuildSummary). These results provide valuable guidelines for the optimal interpretation of proteomic results and the development of fit-for-purpose protocols under different situations.