Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation

Coupling of multiplex isobaric tags for relative and absolute quantitation (iTRAQ) to a sensitive linear ion trap (LTQ) mass spectrometer (MS) is a challenging, but highly promising approach for quantitative high-throughput proteomic profiling. Integration of the advantages of pulsed-Q dissociation (PQD) and collision-activated dissociation (CAD) fragmentation methods into a PQD-CAD hybrid mode, together with PQD optimization and data manipulation with a bioinformatics algorithm, resulted in a robust, sensitive and accurate iTRAQ quantitative proteomic workflow. The workflow was superior to the default PQD setting when profiling the proteome of a gastric cancer cell line, SNU5. Taken together, we established an optimized PQD-CAD hybrid workflow in LTQ-MS for iTRAQ quantitative proteomic profiling that may have wide applications in biological and biomedical research.     

Trade-off between high sensitivity and increased potential for false positive peptide sequence matches using a two-dimensional linear ion trap for tandem mass spectrometry-based proteomics

Two-dimensional linear ion trap mass spectrometers are rapidly becoming the new workhorse instruments for shotgun proteomic analysis of complex peptide mixtures. The objective of this study was to compare the potential for false positive peptide sequence matches between a two-dimensional ion trap instrument and a traditional, three-dimensional ion trap instrument. Through the comparative analysis of a complex protein sample, we found that in order to minimize false positive sequence matches, sequence match scoring criteria must be more stringent for data from the two-dimensional ion trap compared to the three-dimensional ion trap data. Given this increased potential for false positives, we also investigated two potential filtering strategies to reduce the false positive matches for data derived from the two-dimensional ion trap, including trypsin enzyme cleavage filtering, and the addition of peptide physicochemical information as a constraint, specifically peptide isoelectric point. The results described here provide a cautionary tale to researchers, demonstrating the need for careful analysis of MS/MS data from this new class of ion trap instruments, as well as the effectiveness of trypsin enzyme cleavage filtering and peptide pI information in maximizing high confidence protein identifications from this powerful proteomic instrumentation.     

Systematic comparison of a two-dimensional ion trap and a three-dimensional ion trap mass spectrometer in proteomics

The utility and advantages of the recently introduced two-dimensional quadrupole ion trap mass spectrometer in proteomics over the traditional three-dimensional ion trap mass spectrometer have not been systematically characterized. Here we rigorously compared the performance of these two platforms by using over 100,000 tandem mass spectra acquired with identical complex peptide mixtures and acquisition parameters. Specifically we compared four factors that are critical for a successful proteomic study: 1) the number of proteins identified, 2) sequence coverage or the number of peptides identified for every protein, 3) the data base matching SEQUEST X(corr) and S(p) score, and 4) the quality of the fragment ion series of peptides. We found a 4-6-fold increase in the number of peptides and proteins identified on the two-dimensional ion trap mass spectrometer as a direct result of improvement in all the other parameters examined. Interestingly more than 70% of the doubly and triply charged peptides, but not the singly charged peptides, showed better quality of fragmentation spectra on the two-dimensional ion trap. These results highlight specific advantages of the two-dimensional ion trap over the conventional three-dimensional ion traps for protein identification in proteomic experiments.     

Identification of carbonylated proteins from enriched rat skeletal muscle mitochondria using affinity chromatography-stable isotope labeling and tandem mass spectrometry

We describe a strategy for the identification of carbonylated proteins from complex protein mixtures that combines biotin hydrazide labeling of protein carbonyl groups, avidin affinity chromatography, multiplexed iTRAQ reagent stable isotope labeling, and analysis using pulsed Q dissociation (PQD) operation on an LTQ linear ion trap mass spectrometer. This strategy provided the ability to distinguish biotin hydrazide labeled, avidin purified, carbonylated proteins from non-carbonylated background proteins with affinity for the avidin column, derived from a control sample. Applying this strategy to the identification of crudely enriched rat skeletal muscle mitochondrial protein isolates, we generated a catalogue of over 200 carbonylated proteins by virtue of their quantitative enrichment compared to the control sample. The catalogue contains many mitochondrial localized proteins shown to be susceptible to carbonyl modification for the first time, including numerous transmembrane proteins involved in oxidative phosphorylation. Other oxidative modifications (e.g. nitrosylation, hydroxylation) were also identified on many of the carbonylated proteins, providing further evidence of the susceptibility of these proteins to oxidative damage. The results also demonstrate the utility of PQD operation on the LTQ instrument for quantitative analysis of iTRAQ reagent-labeled peptide mixtures, as well as the quantitative reproducibility of the avidin-affinity enrichment method.     

LTQ‐iQuant: A freely available software pipeline for automated and accurate protein quantification of isobaric tagged peptide data from LTQ instruments

Pulsed Q dissociation enables combining LTQ ion trap instruments with isobaric peptide tagging. Unfortunately, this combination lacks a technique which accurately reports protein abundance ratios and is implemented in a freely available, flexible software pipeline. We developed and implemented a technique assigning collective reporter ion intensity‐based weights to each peptide abundance ratio and calculating a protein's weighted average abundance ratio and p‐value. Using an iTRAQ‐labeled standard mixture, we compared our technique's performance to the commercial software MASCOT, finding that it performed better than MASCOT's nonweighted averaging and median peptide ratio techniques, and equal to its weighted averaging technique. We also compared performance of the LTQ‐Orbitrap plus our technique to 4800 MALDI TOF/TOF plus Protein Pilot, by analyzing an iTRAQ‐labeled stem cell lysate. We found highly correlated protein abundance ratios, indicating that the LTQ‐Orbitrap plus our technique yields results comparable to the current standard. We implemented our technique in a freely available, automated software pipeline, called LTQ‐iQuant, which is mzXML‐compatible; supports iTRAQ 4‐plex and 8‐plex LTQ data; and can be modified for and have weights trained to a user's LTQ and other isobaric peptide tagging methods. LTQ‐iQuant should make LTQ instruments and isobaric peptide tagging accessible to more proteomic researchers.     

The fate of b-ions in the two worlds of collision-induced dissociation

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.     

The use of a hybrid linear trap/FT-ICR mass spectrometer for on-line high resolution/high mass accuracy bottom-up sequencing.

In this work we present a hybrid linear trap/Fourier transform ion cyclotron resonance (ICR) mass spectrometer to perform protein sequencing using the bottom-up approach. We demonstrate that incorporation of the linear trap greatly enhances the overall performance of the hybrid system for the study of complex peptide mixtures separated by fast high-performance liquid chromatography gradients. The ability to detect in the linear trap enables employment of automatic gain control to greatly reduce space charging in the ICR cell irregardless of ion flux. Resulting accurate mass measurements of 2 ppm or better using external calibration are achieved for the base peak as well as ions at 2% relative abundance. The linear trap is used to perform ion accumulation and activation prior to detection in the ICR cell which increases the scan rate. The increased duty cycle allows for data-dependent mass analysis of coeluting peptides to be acquired increasing protein sequence coverage without increasing the gradient length. In addition, the linear trap could be used as an ion detection device to perform simultaneous detection of tandem mass spectra with full scan mass spectral detection in the ICR cell resulting in the fastest scan cycles for performing bottom-up sequencing of protein digests. Comparisons of protein sequence coverage are presented for product ion detection in the linear trap and ICR cell.     

蛋白质组研究中离子阱串联质谱数据搜库结果解释方法

基于离子阱串联质谱仪的鸟枪法是一种高通量的蛋白质鉴定方法。得到的数据一般使用软件SEQUEST搜索蛋白质序列数据库,得到肽段鉴定列表以及相应的打分。为了得到蛋白质鉴定列表,还需要进行肽段鉴定结果的过滤和假阳性率的计算,然后根据肽段鉴定结果组装蛋白质列表。这两个问题目前还没有很好地解决。对已有的方法进行总结和比较,可以给搜库结果解释方法的选择提供参考,对数据质量控制方法的改进也有所帮助。     

Electron transfer dissociation of iTRAQ labeled peptide ions

Triply and doubly charged iTRAQ ( isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD and supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/ z 162 yielded the reporter ion at m/ z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents.     

Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR)

We have developed a new and sensitive LC-MS platform, Extended Range Proteomic Analysis (ERPA), which is able to achieve very high sequence coverage and comprehensive characterization of post-translational modifications in complex proteins. This new platform provides advantages of both the top-down and bottom-up proteomic approaches by combining (i) digestion of the protein with an enzyme, such as Lys-C, which cuts less frequently than trypsin, leading to on average a higher molecular weight peptide size, (ii) high-performance LC separation of the resulting fragments, (iii) a new data acquisition strategy using the LTQ-FTMS, a hybrid mass spectrometer that couples a linear ion trap with a Fourier transform ion cyclotron resonance (FTICR) cell, for analysis of peptides in the range of 0.5 to 10 kDa, and (iv) new data analysis methods for assigning large peptide structures and determining the site of attachment of post-translational modifications as well as structural features from the accurate precursor mass together with MS(2) and MS(3) fragmentations. The LC retention of the Lys-C fragments is increased, relative to a tryptic digest, due to the generally greater hydrophobicity of the larger peptides, a result that is particularly important for peptides containing hydrophilic modifications such as glycosylation and phosphorylation. Furthermore, additional positively charged arginine and lysine residues in the Lys-C fragments enhance the sensitivity of the post-translationally modified phospho- and glycopeptides by at least 10-fold relative to tryptic fragments. In typical operation, the FTICR cell provides a survey scan with the high mass resolution (> 100 000) and accurate mass (<2 ppm) to characterize the higher charge-state precursor ions of the larger peptides. In parallel, the linear ion trap provides MS(2) and MS(3) fragmentation spectra, with a scan speed sufficiently fast for on-line LC-MS. Together, these data provide multiple means to determine or enhance the confidence of assignment of large or complicated peptide. Using ERPA, we demonstrate >95% sequence coverage in the analysis of two heavily phosphorylated and glycosylated proteins, beta-casein at the 50 fmole level and the epidermal growth factor receptor (EGFR) at the 1 pmole level. In summary, the combination of digestion strategy, high-performance separation, and the hybrid LTQ-FTMS instrument enables comprehensive characterization of large proteins, including posttranslational modifications.     

Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos

Over the past decade peptide sequencing by collision induced dissociation (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissociation (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.g., in a 2D or 3D ion trap) or at high resolution and high mass accuracy (e.g., an Orbitrap or a ToF). Here, we describe a comprehensive experimental comparison of using ETD, ion-trap CID, and beam type CID (HCD) in combination with either linear ion trap or Orbitrap readout for the large-scale analysis of tryptic peptides. We investigate which combination of fragmentation technique and mass analyzer provides the best performance for the analysis of distinct peptide populations such as N-acetylated, phosphorylated, and tryptic peptides with up to two missed cleavages. We found that HCD provides more peptide identifications than CID and ETD for doubly charged peptides. In terms of Mascot score, ETD FT outperforms the other techniques for peptides with charge states higher than 2. Our data shows that there is a trade-off between spectral quality and speed when using the Orbitrap for fragment ion detection. We conclude that a decision-tree regulated combination of higher-energy collisional dissociation (HCD) and ETD can improve the average Mascot score.     

SALAMI (Spectrum ALignments using high Accuracy Mass and hIgh sensitivity data): how to make the best out of hybrid MS/MS data

We present a software algorithm that combines ion trap and orbitrap product ion spectra acquired in parallel. The hybrid product ion spectra identify more peptides than when using two separate searches for the orbitrap and LTQ data. The program extracts the high-accuracy mass data from the Orbitrap mass analyzer and combines it with the high-sensitivity data analyzed in the LTQ linear ion trap; the m/z values of the high-confidence fragment ions are corrected to orbitrap mass accuracies and the fragment ion intensities are amplified. This approach utilizes the parallel spectrum measurement capabilities of the LTQ-Orbitrap. We present our approach to handling this type of hybrid data, explain our alignment program, and discuss the advantages of the chosen methodology.     

Robust and sensitive iTRAQ quantification on an LTQ Orbitrap mass spectrometer

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.     

Integrated platform for manual and high‐throughput statistical validation of tandem mass spectra

As proteomic data sets increase in size and complexity, the necessity for database‐centric software systems able to organize, compare, and visualize all the proteomic experiments in a lab grows. We recently developed an integrated platform called high‐throughput autonomous proteomic pipeline (HTAPP) for the automated acquisition and processing of quantitative proteomic data, and integration of proteomic results with existing external protein information resources within a lab‐based relational database called PeptideDepot. Here, we introduce the peptide validation software component of this system, which combines relational database‐integrated electronic manual spectral annotation in Java with a new software tool in the R programming language for the generation of logistic regression spectral models from user‐supplied validated data sets and flexible application of these user‐generated models in automated proteomic workflows. This logistic regression spectral model uses both variables computed directly from SEQUEST output in addition to deterministic variables based on expert manual validation criteria of spectral quality. In the case of linear quadrupole ion trap (LTQ) or LTQ‐FTICR LC/MS data, our logistic spectral model outperformed both XCorr (242% more peptides identified on average) and the X!Tandem E‐value (87% more peptides identified on average) at a 1% false discovery rate estimated by decoy database approach.     

Two‐step OFFGEL approach for effective peptide separation compatible with iTRAQ labeling

Shotgun proteomic analyses are increasingly becoming methods of choice for complex samples. The development of effective methods for fractionating peptides to reduce the complexity of the sample before mass analysis is a key point in this strategy. The OFFGEL technology has recently become a tool of choice in proteomic analysis at peptide level. This OFFGEL electrophoresis (OGE) approach allows the in‐solution separation of peptides from various biological sources by isoelectric focusing in highly resolved 24 fractions. It was also demonstrated that OGE technology is a filtering tool for pI‐based validation of peptide identification. As peptide OGE is compatible with iTRAQ labeling, OGE is finding valuable applications in quantitative proteomics as well. The aim of this study is to explain a new 2D‐OGE approach that improves the proteomic coverage of complex mixtures such as colorectal cell line lysates, and which is compatible with iTRAQ labeling.     

The platelet microparticle proteome

Platelet-derived microparticles are the most abundant type of microparticle in human blood and contribute to many biologically significant processes. Here, we report the first proteomic analysis of microparticles generated from activated platelets. Using 1D SDS-PAGE and liquid chromatography coupled to a linear ion trap mass spectrometer, the identification of 578 proteins was accomplished using a minimum of 5 MS/MS detections of at least two different peptides for each protein. These microparticles displayed many proteins intrinsic to and well-characterized on platelets. For example, microparticles in these experiments were found to contain membrane surface proteins including GPIIIa, GPIIb, and P-selectin, as well other platelet proteins such as the chemokines CXCL4, CXCL7, and CCL5. In addition, approximately 380 of the proteins identified were not found in two previous studies of the platelet proteome. Since several of the proteins detected here have been previously implicated in microparticle formation and/or pathological function, it is hoped that this study will help fuel future work concerning the possible role of microparticles in various disease states.     

Halobacterium salinarum NRC-1 PeptideAtlas: toward strategies for targeted proteomics and improved proteome coverage

The relatively small numbers of proteins and fewer possible post-translational modifications in microbes provide a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a PeptideAtlas (PA) covering 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636 000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has highlighted plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore, we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.     

iTRAQ protein quantification: a quality-controlled workflow

Reporter ion-based methods are among the major techniques to quantify peptides and proteins. Two main labels, tandem mass tag (TMT) and iTRAQ, are widely used by the proteomics community. They are, however, often applied as out-of-the-box methods, without thorough quality control. Thus, due to undiscovered limitations of the technique, irrelevant results might be trusted. To address this issue, we here propose a step-by-step quality control of the iTRAQ workflow. From sample preparation to final ratio calculation we provide metrics and techniques assessing the actual effectiveness of iTRAQ quantification as well as a novel method for more reliable protein ratio estimation.     

Low mass cutoff evasion with qz value optimization in ion trap

An ion trap is a powerful analyzer because of its high resolution, high sensitivity, and multistage mass analysis (MSⁿ) capabilities. Multiple fragmentation analysis provides useful information regarding peptide sequence and biomolecular structure; however, this approach is limited by an inherent low mass cutoff (LMCO) derived from collision-induced dissociation (CID). To avoid the LMCO for application of an ion trap to iTRAQ, we optimized the q_z value, which is a parameter that is proportional to the applied fundamental AC radio frequency voltage of a tandem mass spectrometry (MS/MS) event. Considering that many ion trap MS analyses employ CID as the MS/MS method, this method can be a practical one without any instrumental changes.