RESUMO
Targeted mass spectrometry assays for protein quantitation monitor peptide surrogates, which are easily multiplexed to target many peptides in a single assay. However, these assays have generally not taken advantage of sample multiplexing, which allows up to ten analyses to occur in parallel. We present a two-dimensional multiplexing workflow that utilizes synthetic peptides for each protein to prompt the simultaneous quantification of >100 peptides from up to ten mixed sample conditions. We demonstrate that targeted analysis of unfractionated lysates (2 hr) accurately reproduces the quantification of fractionated lysates (72 hr analysis) while obviating the need for peptide detection prior to quantification. We targeted 131 peptides corresponding to 69 proteins across all 60 National Cancer Institute cell lines in biological triplicate, analyzing 180 samples in only 48 hr (the equivalent of 16 min/sample). These data further elucidated a correlation between the expression of key proteins and their cellular response to drug treatment.
Assuntos
Ensaios de Triagem em Larga Escala , Espectrometria de Massas , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteoma , Proteômica/métodos , Antibióticos Antineoplásicos/farmacologia , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Fluxo de TrabalhoRESUMO
Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4-5-fold increases in reporter signal compared to conventional SPS-MS3 approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.
RESUMO
Sample multiplexed quantitative proteomics assays have proved to be a highly versatile means to assay molecular phenotypes. Yet, stochastic precursor selection and precursor coisolation can dramatically reduce the efficiency of data acquisition and quantitative accuracy. To address this, intelligent data acquisition (IDA) strategies have recently been developed to improve instrument efficiency and quantitative accuracy for both discovery and targeted methods. Toward this end, we sought to develop and implement a new real-time spectral library searching (RTLS) workflow that could enable intelligent scan triggering and peak selection within milliseconds of scan acquisition. To ensure ease of use and general applicability, we built an application to read in diverse spectral libraries and file types from both empirical and predicted spectral libraries. We demonstrate that RTLS methods enable improved quantitation of multiplexed samples, particularly with consideration for quantitation from chimeric fragment spectra. We used RTLS to profile proteome responses to small molecule perturbations and were able to quantify up to 15% more significantly regulated proteins in half the gradient time compared to traditional methods. Taken together, the development of RTLS expands the IDA toolbox to improve instrument efficiency and quantitative accuracy for sample multiplexed analyses.
Assuntos
Peptídeos , Proteômica , Proteômica/métodos , Peptídeos/análise , Proteoma/análise , Biblioteca Gênica , Fluxo de Trabalho , Biblioteca de PeptídeosRESUMO
Mass spectrometry (MS)-based proteomics is a powerful technology to globally profile protein abundances, activities, interactions, and modifications. The extreme complexity of proteomics samples, which often contain hundreds of thousands of analytes, necessitates continuous development of MS techniques and instrumentation to improve speed, sensitivity, precision, and accuracy, among other analytical characteristics. Here, we systematically evaluated the Orbitrap Ascend Tribrid mass spectrometer in the context of shotgun proteomics, and we compared its performance to that of the previous generation of Tribrid instrumentsâthe Orbitrap Eclipse. The updated architecture of the Orbitrap Ascend includes a second ion-routing multipole (IRM) in front of the redesigned C-trap/Orbitrap and a new ion funnel that allows gentler ion introduction, among other changes. These modifications in Ascend hardware configuration enabled an increase in parallelizable ion injection time during higher-energy collisional dissociation (HCD) Orbitrap tandem MS (FTMS2) analysis of â¼5 ms. This enhancement was particularly valuable in the analyses of limited sample amounts, where improvements in sensitivity resulted in up to 140% increase in the number of identified tryptic peptides. Further, analysis of phosphorylated peptides enriched from the K562 human cell line yielded up to â¼50% increase in the number of unique phosphopeptides and localized phosphosites. Strikingly, we also observed a â¼2-fold boost in the number of detected N-glycopeptides, likely owing to the improvements in ion transmission and sensitivity. In addition, we performed the multiplexed quantitative proteomics analyses of TMT11-plex labeled HEK293T tryptic peptides and observed 9-14% increase in the number of quantified peptides. In conclusion, the Orbitrap Ascend consistently outperformed its predecessor the Orbitrap Eclipse in various bottom-up proteomic analyses, and we anticipate that it will generate reproducible and in-depth datasets for numerous proteomic applications.
Assuntos
Proteínas , Proteômica , Humanos , Proteômica/métodos , Células HEK293 , Proteínas/química , Espectrometria de Massas em Tandem/métodos , FosfopeptídeosRESUMO
Tandem mass tags (TMT) and tribrid mass spectrometers are a powerful combination for high-throughput proteomics with high quantitative accuracy. Increasingly, this technology is being used to map the effects of drugs on the proteome. However, the depth of proteomic profiling is still limited by sensitivity and speed. The new Orbitrap Ascend mass spectrometer was designed to address these limitations with a combination of hardware and software improvements. We evaluated the performance of the Ascend in multiple contexts including deep proteomic profiling. We found that the Ascend exhibited increased sensitivity, yielding higher signal-to-noise ratios than the Orbitrap Eclipse with shorter injection times. As a result, higher numbers of peptides and proteins were identified and quantified, especially with low sample input. TMT measurements had significantly improved signal-to-noise ratios, improving quantitative precision. In a fractionated 16plex sample that profiled proteomic differences across four human cell lines, the Ascend was able to quantify hundreds more proteins than the Eclipse, many of them low-abundant proteins, and the Ascend was able to quantify >8000 proteins in 30% less instrument time. We used the Ascend to analyze 8881 proteins in HCT116 cancer cells treated with covalent sulfolane/sulfolene inhibitors of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), a phosphorylation-specific peptidyl-prolyl cis-trans isomerase implicated in several cancers. We characterized these PIN1 inhibitors' effects on the proteome and identified discrepancies among the different compounds, which will facilitate a better understanding of the structure-activity relationship of this class of compounds. The Ascend was able to quantify statistically significant, potentially therapeutically relevant changes in proteins that the Eclipse could not detect.
Assuntos
Proteoma , Proteômica , Humanos , Proteoma/metabolismo , Espectrometria de Massas , Células HCT116 , cis-trans-Isomerases , Peptidilprolil Isomerase de Interação com NIMARESUMO
In mass spectrometry-based lipidomics, complex lipid mixtures undergo chromatographic separation, are ionized, and are detected using tandem MS (MSn) to simultaneously quantify and structurally characterize eluting species. The reported structural granularity of these identified lipids is strongly reliant on the analytical techniques leveraged in a study. For example, lipid identifications from traditional collisionally activated data-dependent acquisition experiments are often reported at either species level or molecular species level. Structural resolution of reported lipid identifications is routinely enhanced by integrating both positive and negative mode analyses, requiring two separate runs or polarity switching during a single analysis. MS3+ can further elucidate lipid structure, but the lengthened MS duty cycle can negatively impact analysis depth. Recently, functionality has been introduced on several Orbitrap Tribrid mass spectrometry platforms to identify eluting molecular species on-the-fly. These real-time identifications can be leveraged to trigger downstream MSn to improve structural characterization with lessened impacts on analysis depth. Here, we describe a novel lipidomics real-time library search (RTLS) approach, which utilizes the lipid class of real-time identifications to trigger class-targeted MSn and to improve the structural characterization of phosphotidylcholines, phosphotidylethanolamines, phosphotidylinositols, phosphotidylglycerols, phosphotidylserine, and sphingomyelins in the positive ion mode. Our class-based RTLS method demonstrates improved selectivity compared to the current methodology of triggering MSn in the presence of characteristic ions or neutral losses.
Assuntos
Glicerofosfolipídeos , Esfingomielinas , Glicerofosfolipídeos/análise , Esfingomielinas/análise , Espectrometria de Massas em Tandem/métodos , Íons , Biblioteca GênicaRESUMO
Isobaric tagging facilitates multiplexed experiments that can determine sequences and relative amounts of peptides in biological samples using tandem mass spectrometry (MSn). Limited reporter ion generation limits quantitative accuracy and precision. As reporter ions are susceptible to unintended fragmentation and scattering by high-energy collisions, we activated peptides with IR photons and prevented successive dissociation of generated reporter ions with ion parking, which altogether boosted reporter ion yield by up to 55%. Even so, unintended co-isolation of contaminating peaks in MS2 experiments distorts reporter ion intensities and can distort quantitative information. MS3 experiments address contamination by generating reporter ions via collisional activation (HCD) of one or more peptide product ions rather than the isolated peptide precursor ion. Because HCD performance is related to m/z, activation of multiple synchronously isolated product ions generates less than optimal reporter ion intensities. In this work, we show that using infrared multiphoton dissociation, which is not dependent on m/z, to generate reporter ions from 10 synchronously isolated peptide product ions results in a 2.4-fold increase in reporter ion intensities, significantly enhancing the sensitivity and dynamic range of quantitation via isobaric tagging.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Indicadores e Reagentes , Íons , Peptídeos/química , Espectrometria de Massas em Tandem/métodosRESUMO
Gas-phase fractionation enables better quantitative accuracy, improves signal-to-noise ratios, and increases sensitivity in proteomic analyses. However, traditional gas-phase enrichment, which relies upon a large continuous bin, results in suboptimal enrichment, as most chromatographic separations are not 100% orthogonal relative to the first MS dimension (MS1m/z). As such, ions with similar m/z values tend to elute at the same retention time, which prevents the partitioning of narrow precursor m/z distributions into a few large continuous gas-phase enrichment bins. To overcome this issue, we developed and tested the use of notched isolation waveforms, which simultaneously isolate multiple discrete m/z windows in parallel (e.g., 650-700 m/z and 800-850 m/z). By comparison to a canonical gas-phase fractionation method, notched waveforms do not require bin optimization via in silico digestion or wasteful sample injections to isolate multiple precursor windows. Importantly, the collection of all m/z bins simultaneously using the isolation waveform does not suffer from the sensitivity and duty cycle pitfalls inherent to sequential collection of multiple m/z bins. Applying a notched injection waveform provided consistent enrichment of precursor ions, which resulted in improved proteome depth with greater coverage of low-abundance proteins. Finally, using a reductive dimethyl labeling approach, we show that notched isolation waveforms increase the number of quantified peptides with improved accuracy and precision across a wider dynamic range.
Assuntos
Proteoma , Proteômica , Fracionamento Químico , Íons , PeptídeosRESUMO
The rise of sample multiplexing in quantitative proteomics for the dissection of complex phenotypic comparisons has been advanced by the development of ever more sensitive and robust instrumentation. Here, we evaluated the utility of the Orbitrap Eclipse Tribrid mass spectrometer (advanced quadrupole filter, optimized FTMS scan overhead) and new instrument control software features (Precursor Fit filtering, TurboTMT and Real-time Peptide Search filtering). Multidimensional comparisons of these novel features increased total peptide identifications by 20% for SPS-MS3 methods and 14% for HRMS2 methods. Importantly Real-time Peptide Search filtering enabled a â¼2× throughput improvement for quantification. Across the board, these sensitivity increases were attained without sacrificing quantitative accuracy. New hardware and software features enable more efficient characterization in pursuit of comparative whole proteome insights.
Assuntos
Peptídeos/análise , Proteômica , Espectrometria de MassasRESUMO
Lipid composition can differ widely among organelles and even between leaflets of a membrane. Lipid homeostasis is critical because disequilibrium can have disease outcomes. Despite their importance, mechanisms maintaining lipid homeostasis remain poorly understood. Here, we establish a model system to study the global effects of lipid imbalance. Quantitative lipid profiling was integral to monitor changes to lipid composition and for system validation. Applying global transcriptional and proteomic analyses, a dramatically altered biochemical landscape was revealed from adaptive cells. The resulting composite regulation we term the "membrane stress response" (MSR) confers compensation, not through restoration of lipid composition, but by remodeling the protein homeostasis network. To validate its physiological significance, we analyzed the unfolded protein response (UPR), one facet of the MSR and a key regulator of protein homeostasis. We demonstrate that the UPR maintains protein biogenesis, quality control, and membrane integrity-functions otherwise lethally compromised in lipid dysregulated cells.
Assuntos
Proteínas Ligadas a Lipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Resposta a Proteínas não Dobradas , Homeostase , Proteínas Ligadas a Lipídeos/química , Lipídeos de Membrana/química , Redes e Vias Metabólicas , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse FisiológicoRESUMO
Liquid chromatography (LC) prefractionation is often implemented to increase proteomic coverage; however, while effective, this approach is laborious, requires considerable sample amount, and can be cumbersome. We describe how interfacing a recently described high-field asymmetric waveform ion mobility spectrometry (FAIMS) device between a nanoelectrospray ionization (nanoESI) emitter and an Orbitrap hybrid mass spectrometer (MS) enables the collection of single-shot proteomic data with comparable depth to that of conventional two-dimensional LC approaches. This next generation FAIMS device incorporates improved ion sampling at the ESI-FAIMS interface, increased electric field strength, and a helium-free ion transport gas. With fast internal compensation voltage (CV) stepping (25 ms/transition), multiple unique gas-phase fractions may be analyzed simultaneously over the course of an MS analysis. We have comprehensively demonstrated how this device performs for bottom-up proteomics experiments as well as characterized the effects of peptide charge state, mass loading, analysis time, and additional variables. We also offer recommendations for the number of CVs and which CVs to use for different lengths of experiments. Internal CV stepping experiments increase protein identifications from a single-shot experiment to >8000, from over 100 000 peptide identifications in as little as 5 h. In single-shot 4 h label-free quantitation (LFQ) experiments of a human cell line, we quantified 7818 proteins with FAIMS using intra-analysis CV switching compared to 6809 without FAIMS. Single-shot FAIMS results also compare favorably with LC fractionation experiments. A 6 h single-shot FAIMS experiment generates 8007 protein identifications, while four fractions analyzed for 1.5 h each produce 7776 protein identifications.
Assuntos
Espectrometria de Mobilidade Iônica/instrumentação , Peptídeos/análise , Proteínas/análise , Proteômica/instrumentação , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Linhagem Celular , HumanosRESUMO
Modern ion trap mass spectrometers are capable of collecting up to 60 tandem MS (MS/MS) scans per second, in theory providing acquisition speeds that can sample every eluting peptide precursor presented to the MS system. In practice, however, the precursor sampling capacity enabled by these ultrafast acquisition rates is often underutilized due to a host of reasons (e.g., long injection times and wide analyzer mass ranges). One often overlooked reason for this underutilization is that the instrument exhausts all the peptide features it identifies as suitable for MS/MS fragmentation. Highly abundant features can prevent annotation of lower abundance precursor ions that occupy similar mass-to-charge (m/z) space, which ultimately inhibits the acquisition of an MS/MS event. Here, we present an advanced peak determination (APD) algorithm that uses an iterative approach to annotate densely populated m/z regions to increase the number of peptides sampled during data-dependent LC-MS/MS analyses. The APD algorithm enables nearly full utilization of the sampling capacity of a quadrupole-Orbitrap-linear ion trap MS system, which yields up to a 40% increase in unique peptide identifications from whole cell HeLa lysates (approximately 53â¯000 in a 90 min LC-MS/MS analysis). The APD algorithm maintains improved peptide and protein identifications across several modes of proteomic data acquisition, including varying gradient lengths, different degrees of prefractionation, peptides derived from multiple proteases, and phosphoproteomic analyses. Additionally, the use of APD increases the number of peptides characterized per protein, providing improved protein quantification. In all, the APD algorithm increases the number of detectable peptide features, which maximizes utilization of the high MS/MS capacities and significantly improves sampling depth and identifications in proteomic experiments.
Assuntos
Algoritmos , Fragmentos de Peptídeos/análise , Precursores de Proteínas/análise , Proteoma/análise , Células HeLa , Humanos , Precursores de Proteínas/química , Proteoma/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
As a driver for many biological processes, phosphorylation remains an area of intense research interest. Advances in multiplexed quantitation utilizing isobaric tags (e.g., TMT and iTRAQ) have the potential to create a new paradigm in quantitative proteomics. New instrumentation and software are propelling these multiplexed workflows forward, which results in more accurate, sensitive, and reproducible quantitation across tens of thousands of phosphopeptides. This study assesses the performance of multiplexed quantitative phosphoproteomics on the Orbitrap Fusion mass spectrometer. Utilizing a two-phosphoproteome model of precursor ion interference, we assessed the accuracy of phosphopeptide quantitation across a variety of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows, (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan, (iii) interfering ions generally possessed a different charge state than the target precursor, and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably, these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent, low level background fragmentation. Finally, as a proof-of-concept 10-plex experiment, we compared phosphopeptide levels from five murine brains to five livers. In total, the SPS-MS3 method quantified 38â¯247 phosphopeptides, corresponding to 11â¯000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide, this equates to more than 628â¯000 binary comparisons collected in less than 48 h.
Assuntos
Química Encefálica , Cromatografia Líquida de Alta Pressão/métodos , Fígado/química , Espectrometria de Massas/métodos , Fosfopeptídeos/análise , Proteômica/métodos , Animais , Cromatografia de Fase Reversa/métodos , Masculino , Camundongos , FosforilaçãoRESUMO
We have developed and implemented a sequence identification algorithm (inSeq) that processes tandem mass spectra in real-time using the mass spectrometer's (MS) onboard processors. The inSeq algorithm relies on accurate mass tandem MS data for swift spectral matching with high accuracy. The instant spectral processing technology takes â¼16 ms to execute and provides information to enable autonomous, real-time decision making by the MS system. Using inSeq and its advanced decision tree logic, we demonstrate (i) real-time prediction of peptide elution windows en masse (â¼3 min width, 3,000 targets), (ii) significant improvement of quantitative precision and accuracy (~3x boost in detected protein differences), and (iii) boosted rates of posttranslation modification site localization (90% agreement in real-time vs. offline localization rate and an approximate 25% gain in localized sites). The decision tree logic enabled by inSeq promises to circumvent problems with the conventional data-dependent acquisition paradigm and provides a direct route to streamlined and expedient targeted protein analysis.
Assuntos
Algoritmos , Peptídeos/análise , Proteínas/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Sítios de Ligação , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Bases de Dados de Proteínas , Árvores de Decisões , Humanos , Dados de Sequência Molecular , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteínas/química , Reprodutibilidade dos Testes , Software , Fatores de TempoRESUMO
Multiplexed quantitation via isobaric chemical tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)) has the potential to revolutionize quantitative proteomics. However, until recently the utility of these tags was questionable due to reporter ion ratio distortion resulting from fragmentation of coisolated interfering species. These interfering signals can be negated through additional gas-phase manipulations (e.g., MS/MS/MS (MS3) and proton-transfer reactions (PTR)). These methods, however, have a significant sensitivity penalty. Using isolation waveforms with multiple frequency notches (i.e., synchronous precursor selection, SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby increasing the number of reporter ions in the MS3 spectrum 10-fold over the standard MS3 method (i.e., MultiNotch MS3). By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements. To demonstrate utility, we analyzed biological triplicates of eight colon cancer cell lines using the MultiNotch MS3 method. Across all the replicates we quantified 8,378 proteins in union and 6,168 proteins in common. Taking into account that each of these quantified proteins contains eight distinct cell-line measurements, this data set encompasses 174,704 quantitative ratios each measured in triplicate across the biological replicates. Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments.
Assuntos
Neoplasias do Colo/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/métodos , Células HeLa , Humanos , Íons , Isocitrato Desidrogenase/análise , Isocitrato Desidrogenase/metabolismo , Análise de Componente Principal , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteína Smad4/análise , Proteína Smad4/metabolismo , Espectrometria de Massas em Tandem/instrumentaçãoRESUMO
We describe a mass spectrometry method, QuantMode, which improves accuracy of isobaric tag-based quantification by alleviating the pervasive problem of precursor interference, simultaneous isolation and fragmentation of impurities, through gas-phase purification. QuantMode analysis of a yeast sample 'contaminated' with interfering human peptides showed substantially improved quantitative accuracy compared to a standard scan, with a small loss of spectral identifications. This technique enables large-scale, multiplexed quantitative proteomics using isobaric tagging.
Assuntos
Gases , Proteínas/química , Proteoma , Humanos , Espectrometria de Massas em Tandem/métodosRESUMO
Beam-type collisional activation dissociation (HCD) offers many advantages over resonant excitation collision-activated dissociation, including improved identification of phosphorylated peptides and compatibility with isobaric tag-based quantitation (e.g. tandem mass tag (TMT) and iTRAQ). However, HCD typically requires specially designed and dedicated collision cells. Here we demonstrate that HCD can be performed in the ion injection pathway of a mass spectrometer with a standard atmospheric inlet (iHCD). Testing this method on complex peptide mixtures revealed similar identification rates to collision-activated dissociation (2883 versus 2730 IDs for iHCD/CAD, respectively) and precursor-product-conversion efficiency comparable to that achieved within a dedicated collision cell. Compared with pulsed-q dissociation, a quadrupole ion trap-based method that retains low-mass isobaric tag reporter ions, iHCD yielded isobaric tag for relative and absolute quantification reporter ions 10-fold more intense. This method involves no additional hardware and can theoretically be implemented on any mass spectrometer with an atmospheric inlet.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Técnicas de Cocultura , Células-Tronco Embrionárias/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Oligopeptídeos/química , Proteoma/química , Proteoma/metabolismo , Espectrometria de Massas em Tandem/instrumentação , Leveduras/metabolismoRESUMO
We modified a dual-cell linear ion trap mass spectrometer to perform infrared multiphoton dissociation (IRMPD) in the low-pressure trap of a dual-cell quadrupole linear ion trap (dual-cell QLT) and perform large-scale IRMPD analyses of complex peptide mixtures. Upon optimization of activation parameters (precursor q-value, irradiation time, and photon flux), IRMPD subtly, but significantly, outperforms resonant-excitation collisional-activated dissociation (CAD) for peptides identified at a 1% false-discovery rate (FDR) from a yeast tryptic digest (95% confidence, p = 0.019). We further demonstrate that IRMPD is compatible with the analysis of isobaric-tagged peptides. Using fixed QLT rf amplitude allows for the consistent retention of reporter ions, but necessitates the use of variable IRMPD irradiation times, dependent upon precursor mass to charge (m/z). We show that IRMPD activation parameters can be tuned to allow for effective peptide identification and quantitation simultaneously. We thus conclude that IRMPD performed in a dual-cell ion trap is an effective option for the large-scale analysis of both unmodified and isobaric-tagged peptides.
Assuntos
Raios Infravermelhos , Proteômica , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Bases de Dados Factuais , Espectrometria de Massas , Peptídeos/análise , FótonsRESUMO
Isobaric labeling strategies, such as isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT), have promised to dramatically increase the power of quantitative proteomics. However, when applied to complex mixtures, both the accuracy and precision are undermined by interfering peptide ions that coisolate and cofragment with the target peptide. Additional gas-phase isolation steps, such as proton-transfer ion-ion reactions (PTR) or higher-order MS3 scans, can almost completely eliminate this problem. Unfortunately, these methods come at the expense of decreased acquisition speed and sensitivity. Here we present a method that allows accurate quantification of TMT-labeled peptides at the MS2 level without additional ion purification. Quantification is based on the fragment ion cluster that carries most of the TMT mass balance. In contrast to the use of low m/z reporter ions, the localization of these complement TMT (TMT(C)) ions in the spectrum is precursor-specific; coeluting peptides do not generally affect the measurement of the TMT(C) ion cluster of interest. Unlike the PTR or MS3 strategies, this method can be implemented on a wide range of high-resolution mass spectrometers like the quadrupole Orbitrap instruments (QExactive). A current limitation of the method is that the efficiency of TMT(C) ion formation is affected by both peptide sequence and peptide ion charge state; we discuss potential routes to overcome this problem. Finally, we show that the complement reporter ion approach allows parallelization of multiplexed quantification and therefore holds the potential to multiply the number of distinct peptides that can be quantified in a given time frame.
Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Humanos , Fragmentos de Peptídeos/químicaRESUMO
We describe the first implementation of negative electron-transfer dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer and its application to high-throughput sequencing of peptide anions. NETD, coupled with high pH separations, negative electrospray ionization (ESI), and an NETD compatible version of OMSSA, is part of a complete workflow that includes the formation, interrogation, and sequencing of peptide anions. Together these interlocking pieces facilitated the identification of more than 2000 unique peptides from Saccharomyces cerevisiae representing the most comprehensive analysis of peptide anions by tandem mass spectrometry to date. The same S. cerevisiae samples were interrogated using traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic LC separations, positive ESI, and collision activated dissociation), and the resulting peptide identifications of the different workflows were compared. Due to a decreased flux of peptide anions and a tendency to produce lowly charged precursors, the NETD-based LC-MS/MS workflow was not as sensitive as the positive mode methods. However, the use of NETD readily permits access to underrepresented acidic portions of the proteome by identifying peptides that tend to have lower pI values. As such, NETD improves sequence coverage, filling out the acidic portions of proteins that are often overlooked by the other methods.