On the benefits of acquiring peptide fragment ions at high measured mass accuracy

The advantages and disadvantages of acquiring tandem mass spectra by collision-induced dissociation (CID) of peptides in linear ion trap Fourier-transform hybrid instruments are described. These instruments offer the possibility to transfer fragment ions from the linear ion trap to the FT-based analyzer for analysis with both high resolution and high mass accuracy. In addition, performing CID during the transfer of ions from the linear ion trap (LTQ) to the FT analyzer is also possible in instruments containing an additional collision cell (i.e., the "C-trap" in the LTQ-Orbitrap), resulting in tandem mass spectra over the full m/z range and not limited by the ejection q value of the LTQ. Our results show that these scan modes have lower duty cycles than tandem mass spectra acquired in the LTQ with nominal mass resolution, and typically result in fewer peptide identifications during data-dependent analysis of complex samples. However, the higher measured mass accuracy and resolution provides more specificity and hence provides a lower false positive ratio for the same number of true positives during database search of peptide tandem mass spectra. In addition, the search for modified and unexpected peptides is greatly facilitated with this data acquisition mode. It is therefore concluded that acquisition of tandem mass spectral data with high measured mass accuracy and resolution is a competitive alternative to "classical" data acquisition strategies, especially in situations of complex searches from large databases, searches for modified peptides, or for peptides resulting from unspecific cleavages.     

Precursor ion independent algorithm for top-down shotgun proteomics

We present a precursor ion independent top-down algorithm (PIITA) for use in automated assignment of protein identifications from tandem mass spectra of whole proteins. To acquire the data, we utilize data-dependent acquisition to select protein precursor ions eluting from a C4-based HPLC column for collision induced dissociation in the linear ion trap of an LTQ-Orbitrap mass spectrometer. Gas-phase fractionation is used to increase the number of acquired tandem mass spectra, all of which are recorded in the Orbitrap mass analyzer. To identify proteins, the PIITA algorithm compares deconvoluted, deisotoped, observed tandem mass spectra to all possible theoretical tandem mass spectra for each protein in a genomic sequence database without regard for measured parent ion mass. Only after a protein is identified, is any difference in measured and theoretical precursor mass used to identify and locate post-translation modifications. We demonstrate the application of PIITA to data generated via our wet-lab approach on a Salmonella typhimurium outer membrane extract and compare these results to bottom-up analysis. From these data, we identify 154 proteins by top-down analysis, 73 of which were not identified in a parallel bottom-up analysis. We also identify 201 unique isoforms of these 154 proteins at a false discovery rate (FDR) of <1%.     

Application of a linear ion trap/orbitrap mass spectrometer in metabolite characterization studies: Examination of the human liver microsomal metabolism of the non-tricyclic anti-depressant nefazodone using data-dependent accurate mass measurements

We report herein, facile metabolite identification workflow on the anti-depressant nefazodone, which is derived from accurate mass measurements based on a single run/experimental analysis. A hybrid LTQ/orbitrap mass spectrometer was used to obtain accurate mass full scan MS and MS/MS in a data-dependent fashion to eliminate the reliance on a parent mass list. Initial screening utilized a high mass tolerance ( approximately 10 ppm) to filter the full scan MS data for previously reported nefazodone metabolites. The tight mass tolerance reduces or eliminates background chemical noise, dramatically increasing sensitivity for confirming or eliminating the presence of metabolites as well as isobaric forms. The full scan accurate mass analysis of suspected metabolites can be confirmed or refuted using three primary tools: (1) predictive chemical formula and corresponding mass error analysis, (2) rings-plus-double bonds, and (3) accurate mass product ion spectra of parent and suspected metabolites. Accurate mass characterization of the parent ion structure provided the basis for assessing structural assignment for metabolites. Metabolites were also characterized using parent product ion m/z values to filter all tandem mass spectra for identification of precursor ions yielding similar product ions. Identified metabolite parent masses were subjected to chemical formula calculator based on accurate mass as well as bond saturation. Further analysis of potential nefazodone metabolites was executed using accurate mass product ion spectra. Reported mass measurement errors for all full scan MS and MS/MS spectra was <3 ppm, regardless of relative ion abundance, which enabled the use of predictive software in determining product ion structure. The ability to conduct biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurements, all in a single experimental run, is clearly one of the most attractive features of this methodology.     

Factors that affect ion trap data-dependent MS/MS in proteomics

Quadrupole ion trap scanning parameters for performing bottom-up proteomics in a data-dependent fashion were evaluated on a Finnigan LCQ Deca mass spectrometer. Evaluation of parameters such as the number of averaged full scans, the number of averaged MS/MS scans, and ion injection times were necessary for acquiring high quality MS/MS spectra that yield favorable b and y ion coverage and high correlation to proteins using database searching algorithms. In this study, we demonstrated how the duty cycle of the mass spectrometer affects the number of peptides that can be successfully identified by SEQUEST using a model system of tryptic BSA peptides to mimic a typical complex mixture associated with bottom-up proteomics. The number of averaged scans and the duration of ion accumulation in the trap had a significant effect on the quality of acquired MS/MS spectra. For example, by increasing the ion injection time from 500 ms to 600 ms, peptide HLVDEPQNLIK improved from being improperly identified to being correctly identified with a SEQUEST cross-correlation score of 3.60. As a result of these experiments, we have devised the following set of ion trap parameters for performing bottom-up proteomics analysis in our laboratory: Three averaged full scans, five averaged MS/MS scans, and a maximum ion injection time of 600 ms.     

A generic method to detect electrophilic intermediates using isotopic pattern triggered data-dependent high-resolution accurate mass spectrometry

A need still exists for a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method that can detect broad classes of glutathione (GSH) conjugates and provide characterization of their structures. We now describe the development of a method that multiplexes high-resolution accurate mass analysis with isotope pattern triggered data-dependent product ion scans, for simultaneous detection and structural elucidation of GSH conjugates within a single analysis using a LTQ/Orbitrap. This method was initially developed to detect GSH conjugates generated from incubating 10 microM test compound with pooled human liver microsomes fortified with NADPH-regenerating system and a 2:1 ratio of 5 mM glutathione and [(13)C(2) (15)N-Gly]glutathione. The GSH conjugates were detected by isotope search of mass defect filtered and control subtracted full scan accurate MS data using MetWorks software. This was followed by elucidation of reactive intermediate structures using chemical formulae for both protonated molecules and their product ions from accurate masses in a single analysis. The mass accuracies measured for the precursor and product ions by the Orbitrap were <2 ppm in external mass calibration mode. Successful detection and characterization of GSH conjugates of acetaminophen, tienilic acid, clozapine, ticlopidine and mifepristone validated this method. In each case, the detected GSH conjugates were within the top five hits by isotope search. This method also has a broader detection capability since it is independent of the collision-induced dissociation behavior of the GSH conjugates. Furthermore, this method is amenable to a broad class of reactive intermediate trapping agents as exemplified by the simultaneous detection and structural elucidation of the cyano-N-methylene iminium ion conjugates of verapamil and its O-desmethyl metabolites, which we report for the first time. In addition to the chemically tagged reactive intermediates, this method also provides information on stable metabolites from the full scan accurate MS data.     

Metabolite identification by data-dependent accurate mass spectrometric analysis at resolving power of 60,000 in external calibration mode using an LTQ/Orbitrap

Performance evaluation of accurate mass measurement by the LTQ/Orbitrap, at a resolving power of 60,000 and in external calibration mode, indicated that the Orbitrap is capable of providing high mass accuracy of <2 ppm for over 24 h post-calibration. This, together with limited trade-off between sensitivity and resolving power plus a wide dynamic range for mass accuracy, suggested that the LTQ/Orbitrap is an ideal analytical tool for structural elucidation of metabolites. The application of the LTQ/Orbitrap to identification of human liver microsomal metabolites of carvedilol was evaluated, using parent mass list triggered data-dependent multiple-stage accurate mass analysis, at a resolving power of 60,000 in external calibration mode. A metabolite identification workflow was developed to utilize chemical formulas from high-resolution accurate mass measurements to confirm structures of product ions of a drug proposed by Mass Frontier, illustrated by identification of structures used to establish lineage of product ions of carvedilol, which later served as a template for identification of its metabolites. A total of 58 in vitro metabolites of carvedilol were detected using 5-ppm mass tolerance filters for theoretical m/z of protonated molecules of predicted metabolites in addition to product ions and neutral mass losses diagnostic of carvedilol. The chemical formulas with unsaturation numbers calculated from the accurate m/z of precursor and product ions can be used to assign, with a high degree of confidence, the structures of metabolites and the sites of metabolism. The mass accuracies obtained for all full scan MS and MSn spectra were <2 ppm. The majority of the metabolites identified agreed with those previously reported except for those that have not been reported before. For example, several glutathione conjugates of carvedilol were reported for the first time, which may explain the reported hepatotoxicity during clinical trials and recent clinical use.     

Rapid characterization of covalent modifications to rat brain mitochondrial proteins after ex vivo exposure to 4-hydroxy-2-nonenal by liquid chromatography-tandem mass spectrometry using data-dependent and neutral loss-driven MS3 acquisition

The modification of mitochondrial proteins enriched from rat forebrain by the major lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was investigated using high performance liquid chromatography (HPLC) and tandem mass spectrometry. Subcellular fractionation in conjunction with a 'shotgun-based' approach that involved both conventional data-dependent and neutral loss (NL)-driven MS(3) data acquisition on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT) was utilized. Using a relatively rapid linear HPLC gradient (1 h) for complex mixture analysis, 24 sites of HNE modification on 15 unique proteins were identified which corresponded exclusively to Michael adduct formation on histidine residues. Since a number of HNE-modified peptides produced a predominant HNE NL fragment-ion signal upon collision-induced dissociation (CID), NL-driven MS(3) data-dependent acquisition was a valuable method to enhance fragmentation information for these particular modified peptides. Of the 24 HNE modification sites identified, approximately 25% were determined from the MS(3) spectra alone. We envision the reported methodology as an efficient screening approach for HNE modification site selectivity that could ultimately provide a foundation for the development of targeted schemes for the characterization of in vivo HNE-protein adducts.     

Quantitation of small molecules using high‐resolution accurate mass spectrometers – a different approach for analysis of biological samples

The quantitative capabilities of a linear ion trap high‐resolution mass spectrometer (LTQ‐Orbitrap™) were investigated using full scan mode bracketing the m/z range of the ions of interest and utilizing a mass resolution (mass/FWHM) of 15000. Extracted ion chromatograms using a mass window of ±5–10 mmu centering on the theoretical m/z of each analyte were generated and used for quantitation. The quantitative performance of the LTQ‐Orbitrap™ was compared with that of a triple quadrupole (API 4000) operating using selected reaction monitoring (SRM) detection. Comparable assay precision, accuracy, linearity and sensitivity were observed for both approaches. The concentrations of actual study samples from 15 Merck drug candidates reported by the two methods were statistically equivalent. Unlike SRM being a tandem mass spectrometric (MS/MS)‐based detection method, a high resolution mass spectrometer operated in full scan does not need MS/MS optimization. This approach not only provides quantitative results for compounds of interest, but also will afford data on other analytes present in the sample. An example of the identification of a major circulating metabolite for a preclinical development study is demonstrated. Copyright © 2009 John Wiley & Sons, Ltd.     

The effects of mass accuracy,data acquisition speed,and search algorithm choice on peptide identification rates in phosphoproteomics

Proteomic analyses via tandem mass spectrometry have been greatly enhanced by the recent development of fast, highly accurate instrumentation. However, successful application of these developments to high-throughput experiments requires careful optimization of many variables which adversely affect each other, such as mass accuracy and data collection speed. We examined the performance of three shotgun-style acquisition methods ranging in their data collection speed and use of mass accuracy in identifying proteins from yeast-derived complex peptide and phosphopeptide-enriched mixtures. We find that the combination of highly accurate precursor masses generated from one survey scan in the FT-ICR cell, coupled with ten data-dependent tandem MS scans in a lower-resolution linear ion trap, provides more identifications in both mixtures than the other examined methods. For phosphopeptide identifications in particular, this method identified over twice as many unique phosphopeptides as the second-ranked, lower-resolution method from triplicate 90-min analyses (744 ± 50 vs. 308 ± 50, respectively). We also examined the performance of four popular peptide assignment algorithms (Mascot, Sequest, OMSSA, and Tandem) in analyzing the results from both high-and low-resolution data. When compared in the context of a false positive rate of approximately 1%, the performance differences between algorithms were much larger for phosphopeptide analyses than for an unenriched, complex mixture. Based upon these findings, acquisition speed, mass accuracy, and the choice of assignment algorithm all largely affect the number of peptides and proteins identified in high-throughput studies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

Automatic recalibration and processing of tandem mass spectra using formula annotation

High accuracy, high resolution tandem mass spectrometry (MS/MS) is becoming more common in analytical applications, yet databases of these spectra remain limited. Databases require good quality spectra with sufficient compound information, but processing, calibration, noise reduction and retrieval of compound information are time‐consuming tasks that prevent many contributions. We present a comprehensive workflow for the automatic processing of MS/MS using formula annotation for recalibration and cleanup to generate high quality spectra of standard compounds for upload to MassBank ( www.massbank.jp ). Compound information is retrieved via Internet services. Reference standards of 70 pesticides were measured at various collision energies on an LTQ‐Orbitrap XL to develop and evaluate the workflow. A total of 944 resulting spectra are now available on MassBank. Evidence of nitrogen adduct formation during MS/MS fragmentation processes was found, highlighting the benefits high accuracy MS/MS offers for spectral interpretation. A database of recalibrated, cleaned‐up spectra resulted in the most correct spectra ranked in first place, regardless of whether the search spectra were recalibrated or not, whereas the average rank of the correct molecular formula was improved from 2.55 (uncalibrated) to 1.53 when using recalibrated MS/MS data. The workflow is available as an R package RMassBank capable of generating MassBank records from raw MS and MS/MS data and can be adjusted to process data acquired with different settings and instruments. This workflow is a vital step towards addressing the need for more high quality, high accuracy MS/MS spectra in spectral databases and provides important information for spectral interpretation. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of a gel-separated unknown glycoprotein by liquid chromatography/multistage tandem mass spectrometry: analysis of rat brain Thy-1 separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

We developed an efficient and convenient strategy for protein identification and glycosylation analysis of a small amount of unknown glycoprotein in a biological sample. The procedure involves isolation of proteins by electrophoresis and mass spectrometric peptide/glycopeptide mapping by LC/ion trap mass spectrometer. For the complete glycosylation analysis, proteins were extracted in intact form from the gel, and proteinase-digested glycoproteins were then subjected to LC/multistage tandem MS (MSn) incorporating a full mass scan, in-source collision-induced dissociation (CID), and data-dependent MSn. The glycopeptides were localized in the peptide/glycopeptide map by using oxonium ions such as HexNAc+ and NeuAc+, generated by in-source CID, and neutral loss by CID-MS/MS. We conducted the search analysis for the glycopeptide identification using search parameters containing a possible glycosylation at the Asn residue with N-acetylglucosamine (203 Da). We were able to identify the glycopeptides resulting from predictable digestion with proteinase. The glycopeptides caused by irregular cleavages were not identified by the database search analysis, but their elution positions were localized using oxonium ions produced by in-source CID, and neutral loss by the data-dependent MSn. Then, all glycopeptides could be identified based on the product ion spectra which were sorted from data-dependent CID-MSn spectra acquired around localized positions. Using this strategy, we successfully elucidated site-specific glycosylation of Thy-1, glycosylphosphatidylinositol (GPI)-anchored proteins glycosylated at Asn23, 74, and 98, and at Cys111. High-mannose-type, complex-type, and hybrid-type oligosaccharides were all found to be attached to Asn23, 74 and 98, and four GPI structures could be characterized. Our method is simple, rapid and useful for the characterization of unknown glycoproteins in a complex mixture of proteins.     

Reliable detection of milk allergens in food using a high-resolution, stand-alone mass spectrometer

Reliable methods are needed for detection of allergenic milk proteins in complex food matrixes. The feasibility of an LC/high-resolution MS method for the analysis of milk proteins in a thermally processed model food (incurred cookies) and in white wine spiked, respectively, with milk powder and caseinate is described. Detection of milk proteins was based on the identification of unique peptides in the tryptic digests of cookie/wine extracts using an RP-HPLC separation coupled to an Exactive nonhybrid mass spectrometer using Orbitrap technology. The extremely high mass accuracy and resolution provided by the Orbitrap analyzer allowed a fast preliminary identification of four previously proposed peptide markers of caseins using only accurate values of the m/z of their ions. No interference was observed, despite the complexity of the analyzed matrixes. Moreover, the availability of a high- energy, collisionally activated dissociation cell integrated in the mass spectrometer enabled acquisition of peptide MS/MS-like spectra through post-source fragmentation. Confirmation of peptide marker identity could then be achieved by a comparison between experimental and predicted product ions. The described method shows the great potential of Orbitrap MS as a reliable technique in the field of protein allergen detection once the peptide markers are identified.     

Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry

Tomato (Lycopersicon esculentum Mill.) is the second most important fruit crop worldwide. Tomatoes are a key component in the Mediterranean diet, which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we use a combination of mass spectrometry (MS) techniques with negative ion detection, liquid chromatography/electrospray ionization linear ion trap quadrupole‐Orbitrap‐mass spectrometry (LC/ESI‐LTQ‐Orbitrap‐MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) on a triple quadrupole, for the identification of the constituents of tomato samples. First, we tested for the presence of polyphenolic compounds through generic MS/MS experiments such as neutral loss and precursor ion scans on the triple quadrupole system. Confirmation of the compounds previously identified was accomplished by injection into the high‐resolution system (LTQ‐Orbitrap) using accurate mass measurements in MS, MS² and MS³ modes. In this way, 38 compounds were identified in tomato samples with very good mass accuracy (<2 mDa), three of them, as far as we know, not previously reported in tomato samples. Copyright © 2010 John Wiley & Sons, Ltd.     

New and automated MS<Superscript>n</Superscript> approaches for top-down identification of modified proteins

An automated top-down approach including data-dependent MS(3) experiment for protein identification/characterization is described. A mixture of wild-type yeast proteins has been separated on-line using reverse-phase liquid chromatography and introduced into a hybrid linear ion trap (LTQ) Fourier transform ion cylclotron resonance (FTICR) mass spectrometer, where the most abundant molecular ions were automatically isolated and fragmented. The MS(2) spectra were interpreted by an automated algorithm and the resulting fragment mass values were uploaded to the ProSight PTM search engine to identify three yeast proteins, two of which were found to be modified. Subsequent MS(3) analyses pinpointed the location of these modifications. In addition, data-dependent MS(3) experiments were performed on standard proteins and wild-type yeast proteins using the stand alone linear trap mass spectrometer. Initially, the most abundant molecular ions underwent collisionally activated dissociation, followed by data-dependent dissociation of only those MS(2) fragment ions for which a charge state could be automatically determined. The resulting spectra were processed to identify amino acid sequence tags in a robust fashion. New hybrid search modes utilized the MS(3) sequence tag and the absolute mass values of the MS(2) fragment ions to collectively provide unambiguous identification of the standard and wild-type yeast proteins from custom databases harboring a large number of post-translational modifications populated in a combinatorial fashion.     

Metabolite identification in rat brain microdialysates by direct infusion nanoelectrospray ionization after desalting on a ZipTip and LTQ/Orbitrap mass spectrometry

Analyzing brain microdialysate samples by mass spectrometry is challenging due to the high salt content of the artificial cerebral spinal fluid (aCSF), low analyte concentrations and small sample volumes collected. A drug and its major metabolites can be examined in brain microdialysates by targeted approaches such as selected reaction monitoring (SRM) which provides selectivity and high sensitivity. However, this approach is not well suited for metabolite profiling in the brain which aims to determine biotransformation pathways. Identifying minor metabolites, or metabolites that arise from brain metabolism, remains a challenge and, for a drug in early discovery, identification of metabolites present in the brain can provide useful information for understanding the pharmacological activity and potential toxicological liabilities of the drug. A method is described here for rapid metabolite profiling in brain microdialysates that involves sample clean‐up using C18 ZipTips to remove salts followed by direct infusion nanoelectrospray with an LTQ/Orbitrap mass spectrometer using real‐time internal recalibration. Full scan mass spectra acquired at high resolving power (100 K at m/z 400) were examined manually and with mass defect filtering. Metabolite identification was aided by sub‐parts‐per‐million mass accuracy and structural characterization was accomplished by tandem mass spectrometry (MS/MS) experiments in the Orbitrap or LTQ depending on the abundance of the metabolite. Using this approach, brain microdialysate samples from rats dosed with one of four CNS drugs (imipramine, reboxetine, citalopram or trazodone) were examined for metabolites. For each drug investigated, metabolites, some of which not previously reported in rat brain, were identified and characterized. Copyright © 2009 John Wiley & Sons, Ltd.     

A novel approach for identification and characterization of glycoproteins using a hybrid linear ion trap/FT-ICR mass spectrometer

Combining source collision-induced dissociation (CID) and tandem mass spectral acquisition in a pseudo-MS(3) experiment using a linear ion trap results in a highly selective and sensitive approach to identifying glycopeptide elution from a protein digest. The increased sensitivity is partially attributed to the nonselective nature of source CID, which allows simultaneous activation of all charge states and coeluting glycoforms generating greater ion abundance for the mass-to-charge (m/z) 204 and/or 366 oxonium ions. Unlike source CID alone, a pseudo-MS(3) approach adds selectivity while improving sensitivity by eliminating chemical noise during the tandem mass spectral acquisition of the oxonium ions in the linear ion trap. Performing the experiments in the hybrid linear ion trap/Fourier transform-ion cyclotron resonance (FT-ICR) enables subsequent high-resolution/high-mass accuracy full-scan mass spectra (MS) and parallel acquisition of MS/MS in the linear ion trap to be completed in 2 s directly following the pseudo-MS(3) scan to collate identification and characterization of glycopeptides in one experimental scan cycle. Analysis of bovine fetuin digest using the combined pseudo-MS(3), high-resolution MS, and data-dependent MS/MS events resulted in identification of four N-linked and two O-linked glycopeptides without enzymatic cleavage of the sugar moiety or release of the sialic acids before analysis. In addition, over 95% of the total protein sequence was identified in one analytical run.     

Two‐injection workflow for a liquid chromatography/LTQ‐Orbitrap system to complete in vivo biotransformation characterization: demonstration with buspirone metabolite identification

The relatively high background matrix in in vivo samples typically poses difficulties in drug metabolite identification, and causes repeated analytical runs on unit resolution liquid chromatography/mass spectrometry (LC/MS) systems before the completion of biotransformation characterization. Ballpark parameter settings for the LTQ‐Orbitrap are reported herein that enable complete in vivo metabolite identification within two HPLC/MS injections on the hybrid LTQ‐Orbitrap data collection system. By setting the FT survey full scan at 60K resolution to trigger five dependent LTQ MS² scans, and proper parameters of Repeat Duration, Exclusion Duration and Repeat Count for the first run (exploratory), the Orbitrap achieved the optimal parallel data acquisition capability and collected maximum number of product ion scans. Biotransformation knowledge based prediction played the key role in exact mass ion extraction and multiple mass defect filtration when the initial data was processed. Meanwhile, product ion extraction and neutral loss extraction of the initial dependent data provided additional bonus in identifying metabolites. With updated parent mass list and the data‐dependent setting to let only the ions on the parent mass list trigger dependent scans, the second run (confirmatory) ensures that all precursor ions of identified metabolites trigger not only dependent product ion scans, but also at or close to the highest concentration of the eluted metabolite peaks. This workflow has been developed for metabolite identification of in vivo or ADME studies, of which the samples typically contain a high level of complex matrix. However, due to the proprietary nature of the in vivo studies, this workflow is presented herein with in vitro buspirone sample incubated with human liver microsomes (HLM). The major HLM‐mediated biotransformation on buspirone was identified as oxidation or hydroxylation since five mono‐ (+16 Da), seven di‐ (+32 Da) and at least three tri‐oxygenated (+48 Da) metabolites were identified. Besides the metabolites 1‐pyrimidinylpiperazine (1‐PP) and hydroxylated 1‐PP that formed by N‐dealkylation, a new metabolite M308 was identified as the result of a second N‐dealkylation of the pyrimidine unit. Two new metabolites containing the 8‐butyl‐8‐azaspiro[4,5]decane‐7,9‐dione partial structure, M240 and M254, were also identified that were formed apparently due to the first N‐dealkylation of the 1‐PP moiety. Copyright © 2009 John Wiley & Sons, Ltd.     

Factorial Experimental Designs Elucidate Significant Variables Affecting Data Acquisition on a Quadrupole Orbitrap Mass Spectrometer

Instrument parameter values for a quadrupole Orbitrap mass spectrometer were optimized for performing global proteomic analyses. Fourteen factors were evaluated for their influence on data-dependent acquisition with an emphasis on both the rate of sequencing and spectral quality by maximizing two individually tested response variables (unique peptides and protein groups). Of the 14 factors, 12 factors were assigned significant contrast values (P?<?0.05) for both response variables. Fundamentally, when optimizing parameters, a balance between spectral quality and duty cycle needs to be reached in order to maximize proteome coverage. This is especially true when using a data-dependent approach for sequencing complex proteomes. For example, maximum ion injection time, automatic gain control settings, and minimum threshold settings for triggering MS/MS isolation and activation all heavily influence ion signal, the number of spectra collected, and spectral quality. To better assess the effect these parameters have on data acquisition, all MS/MS data were parsed according to ion abundance by calculating the percent of the AGC target reached for each MS/MS event and then compared with successful peptide-spectrum matches. This proved to be an effective approach for understanding the effect of ion abundance on successful peptide-spectrum matches and establishing minimum ion abundance thresholds for triggering MS/MS isolation and activation.

Comparison of Data Acquisition Strategies on Quadrupole Ion Trap Instrumentation for Shotgun Proteomics

The most common data collection in shotgun proteomics is via data-dependent acquisition (DDA), a process driven by an automated instrument control routine that directs MS/MS acquisition from the highest abundant signals to the lowest. An alternative to DDA is data-independent acquisition (DIA), a process in which a specified range in m/z is fragmented without regard to prioritization of a precursor ion or its relative abundance in the mass spectrum, thus potentially offering a more comprehensive analysis of peptides than DDA. In this work, we evaluate both DDA and DIA on three different linear ion trap instruments: an LTQ, an LTQ modified with an electrodynamic ion funnel, and an LTQ Velos. These instruments represent both older (LTQ) and newer (LTQ Velos) ion trap designs (i.e., linear versus dual ion traps, respectively), and allow direct comparison of peptide identifications using both DDA and DIA analysis. Further, as the LTQ Velos has an enhanced “S-lens” ion guide to improve ion flux, we found it logical to determine if the former LTQ model could be leveraged by improving sensitivity by modifying with an electrodynamic ion guide of significantly different design to the S-lens. We find that the ion funnel enabled LTQ identifies more proteins in the insoluble fraction of a yeast lysate than the other two instruments in DIA mode, whereas the faster scanning LTQ Velos performs better in DDA mode. We explore reasons for these results, including differences in scan speed, source ion optics, and linear ion trap design. Graphical Abstract

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