RESUMO
The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.
Assuntos
Glicoproteínas , Polissacarídeos , Glicosilação , Glicoproteínas/metabolismo , Espectrometria de Massas/métodos , Polissacarídeos/metabolismoRESUMO
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
Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography-tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2 method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2 data acquisition and more than doubling the number of proteoforms identified in previous studies.
Assuntos
Espectrometria de Mobilidade Iônica , Soro , Humanos , Espectrometria de Mobilidade Iônica/métodos , Soro/química , Proteômica/métodos , Proteínas/análise , Espectrometria de Massas/métodosRESUMO
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
The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0-30 kDa mass range extracted from whole cell or tissue lysates. Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography-tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.
Assuntos
Espectrometria de Mobilidade Iônica , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Proteômica/métodos , Proteínas/análise , Cromatografia Líquida , Escherichia coli/químicaRESUMO
Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker-modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.
Assuntos
Escherichia coli , Peptídeos , Peptídeos/química , Proteínas/química , Espectrometria de Massas/métodos , Reagentes de Ligações Cruzadas/químicaRESUMO
Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.
Assuntos
Proteínas de Choque Térmico HSP70 , Proteínas de Saccharomyces cerevisiae , Humanos , Ligação Proteica , Proteínas de Choque Térmico HSP70/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Processamento de Proteína Pós-Traducional , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP90/metabolismoRESUMO
Immune monitoring in cancer immunotherapy involves screening CD8+ T-cell responses against neoantigens, the tumor-specific peptides presented by Major histocompatibility complex Class I (MHCI) on the cell surface. High-throughput immune monitoring requires methods to produce and characterize small quantities of thousands of MHCI-peptide complexes that may be tested for a patient's T-cell response. MHCI synthesis has been achieved using a photocleavable peptide that is exchanged by the neoantigen; however, assays that measure peptide exchange currently disassemble the complex prior to analysisâprecluding direct molecular characterization. Here, we use native mass spectrometry (MS) to profile intact recombinant MHCI complexes and directly measure peptide exchange. Coupled with size-exclusion chromatography or capillary-zone electrophoresis, the assay identified all tested human leukocyte antigen (HLA)/peptide combinations in the nanomole to picomole range with minimal run time, reconciling the synthetic and analytical requirements of MHCI-peptide screening with the downstream T-cell assays. We further show that the assay can be "multiplexed" by measuring exchange of multiple peptides simultaneously and also enables calculation of Vc50, a measure of gas-phase stability. Additionally, MHCI complexes were fragmented by top-down sequencing, demonstrating that the intact complex, peptide sequence, and their binding affinity can be determined in a single analysis. This screening tool for MHCI-neoantigen complexes represents a step toward the application of state-of-the-art MS technology in translational settings. Not only is this assay already informing on the viability of immunotherapy in practice, the platform also holds promise to inspire novel MS readouts for increasingly complex biomolecules used in the diagnosis and treatment of disease.
Assuntos
Antígenos de Histocompatibilidade Classe I , Peptídeos , Humanos , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/química , Espectrometria de Massas , Antígenos HLA , Antígenos de NeoplasiasRESUMO
Complete LC-MS-based protein primary sequence characterization requires measurement of intact protein profiles under denaturing and/or reducing conditions. To address issues of protein overcharging of unstructured proteins under acidic, denaturing conditions and sample heterogeneity (macro- and micro-scales) which often confound denaturing intact mass analysis of a wide variety of protein samples, we propose the use of broadband isolation of entire charge state distributions of intact proteins followed by ion-ion proton transfer charge reduction, which we have termed "full scan PTCR" (fsPTCR). Using rapid denaturing size exclusion chromatography coupled to fsPTCR-Orbitrap MS and time-resolved deconvolution data analysis, we demonstrate a strategy for method optimization, leading to significant analytical advantages over conventional MS1. Denaturing analysis of the flexible bacterial translation initiation factor 2 (91 kDa) using fsPTCR reduced overcharging and showed an 11-fold gain in S/N compared to conventional MS1. Analysis by fsPTCR-MS of the microheterogeneous glycoprotein fetuin revealed twice as many proteoforms as MS1 (112 vs 56). In a macroheterogeneous mixture of proteins ranging from 14 to 148 kDa, fsPTCR provided more than 10-fold increased sensitivity and quantitative accuracy for diluted bovine serum albumin (66 kDa). Finally, our analysis shows that collisional gas pressure is a key parameter which can be utilized during fsPTCR to retain or remove larger proteins from acquired spectra.
Assuntos
Prótons , Soroalbumina Bovina , Sequência de Aminoácidos , Cromatografia Líquida/métodos , Espectrometria de Massas , Soroalbumina Bovina/químicaRESUMO
In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed scMS requires high ion injection times and high-resolution spectra to quantify the single-cell signal; however, the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time-intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and present a new acquisition strategy termed RETICLE (RTS enhanced quant of single cell spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. We show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at an MS2 injection time of 750 ms using a 2 h gradient.
Assuntos
Proteoma , Proteômica , Espectrometria de Massas , PeptídeosRESUMO
Transmembrane α-helical domains of membrane proteins tend to remain structured in the gas phase, presenting a challenge for efficient electron capture/transfer dissociation during top-down dissociation mass spectrometry (MS) experiments. In this study, we compare results from different dissociation modes on a modern Orbitrap platform applied to a model integral membrane protein containing two transmembrane helices, the c-subunit of the Fo domain of the chloroplast ATP synthase. Using commercially available options, we compare collisionally activated dissociation (CAD) with the related variant higher-energy collisional dissociation (HCD) and with electron transfer dissociation (ETD). HCD performed better than CAD and ETD. A combined method utilizing both ETD and HCD (EThcD) demonstrates significant synergy over HCD or ETD alone, representing a robust option analogous to activated ion electron capture dissociation, whereby an infrared laser was used to heat the protein ion alongside electron bombardment. Ultraviolet photodissociation at 213 nm displays at least three backbone dissociation mechanisms and covered nearly 100% of backbone bonds, suggesting significant potential for this technique.
Assuntos
ATPases de Cloroplastos Translocadoras de Prótons/química , Espectrometria de Massas/métodos , Proteínas de Membrana/química , ATPases de Cloroplastos Translocadoras de Prótons/isolamento & purificação , Transporte de Elétrons , Espectrometria de Massas/instrumentação , Proteínas de Membrana/isolamento & purificação , Processos Fotoquímicos , Conformação Proteica em alfa-Hélice , Raios UltravioletaRESUMO
Obtaining extensive sequencing of an intact protein is essential in order to simultaneously determine both the nature and exact localization of chemical and genetic modifications which distinguish different proteoforms arising from the same gene. To effectively achieve such characterization, it is necessary to take advantage of the analytical potential offered by the top-down mass spectrometry approach to protein sequence analysis. However, as a protein increases in size, its gas-phase dissociation produces overlapping, low signal-to-noise fragments. The application of advanced ion dissociation techniques such as electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD) can improve the sequencing results compared to slow-heating techniques such as collisional dissociation; nonetheless, even ETD- and UVPD-based approaches have thus far fallen short in their capacity to reliably enable extensive sequencing of proteoforms ≥30 kDa. To overcome this issue, we have applied proton transfer charge reduction (PTCR) to limit signal overlap in tandem mass spectra (MS2) produced by ETD (alone or with supplemental ion activation, EThcD). Compared to conventional MS2 experiments, following ETD/EThcD MS2 with PTCR MS3 prior to m/z analysis of deprotonated product ions in the Orbitrap mass analyzer proved beneficial for the identification of additional large protein fragments (≥10 kDa), thus improving the overall sequencing and in particular the coverage of the central portion of all four analyzed proteins spanning from 29 to 56 kDa. Specifically, PTCR-based data acquisition led to 39% sequence coverage for the 56 kDa glutamate dehydrogenase, which was further increased to 44% by combining fragments obtained via HCD followed by PTCR MS3.
RESUMO
Field asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.
Assuntos
Espectrometria de Mobilidade Iônica , Proteômica , Espectrometria de Massas , ProteínasRESUMO
Performing large-scale plasma proteome profiling is challenging due to limitations imposed by lengthy preparation and instrument time. We present a fully automated multiplexed proteome profiling platform (AutoMP3) using the Hamilton Vantage liquid handling robot capable of preparing hundreds to thousands of samples. To maximize protein depth in single-shot runs, we combined 16-plex Tandem Mass Tags (TMTpro) with high-field asymmetric waveform ion mobility spectrometry (FAIMS Pro) and real-time search (RTS). We quantified over 40 proteins/min/sample, doubling the previously published rates. We applied AutoMP3 to investigate the naked mole-rat plasma proteome both as a function of the circadian cycle and in response to ultraviolet (UV) treatment. In keeping with the lack of synchronized circadian rhythms in naked mole-rats, we find few circadian patterns in plasma proteins over the course of 48 h. Furthermore, we quantify many disparate changes between mice and naked mole-rats at both 48 h and one week after UV exposure. These species differences in plasma protein temporal responses could contribute to the pronounced cancer resistance observed in naked mole-rats. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD022891.
Assuntos
Espectrometria de Mobilidade Iônica , Proteômica , Animais , Proteínas Reguladoras de Apoptose , Espectrometria de Massas , Camundongos , Ratos-Toupeira , ProteomaRESUMO
Non-target screening (NTS) based on the combination of liquid chromatography coupled to high-resolution mass spectrometry has become the key method to identify organic micro-pollutants (OMPs) in water samples. However, a large number of compounds remains unidentified with current NTS approaches due to poor quality fragmentation spectra generated by suboptimal fragmentation methods. Here, the potential of the alternative fragmentation technique ultraviolet photodissociation (UVPD) to improve identification of OMPs in water samples was investigated. A diverse set of water-relevant OMPs was selected based on k-means clustering and unsupervised artificial neural networks. The selected OMPs were analyzed using an Orbitrap Fusion Lumos equipped with UVPD. Therewith, information-rich MS2 fragmentation spectra of compounds that fragment poorly with higher-energy collisional dissociation (HCD) could be attained. Development of an R-based data analysis workflow and user interface facilitated the characterization and comparison of HCD and UVPD fragmentation patterns. UVPD and HCD generated both unique and common fragments, demonstrating that some fragmentation pathways are specific to the respective fragmentation method, while others seem more generic. Application of UVPD fragmentation to the analysis of surface water enabled OMP identification using existing HCD spectral libraries. However, high-throughput applications still require optimization of informatics workflows and spectral libraries tailored to UVPD.
Assuntos
Quimioinformática/métodos , Compostos Orgânicos/análise , Fotoquímica/métodos , Raios Ultravioleta , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Cromatografia Líquida , Análise por Conglomerados , Interpretação Estatística de Dados , Monitoramento Ambiental/métodos , Espectrometria de Massas/métodos , Modelos Estatísticos , Redes Neurais de Computação , Linguagens de Programação , Padrões de Referência , Software , Água/análise , Abastecimento de ÁguaRESUMO
Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.
Assuntos
Lipídeos/análise , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Metaboloma , Proteoma/análise , Humanos , LigantesRESUMO
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
Intact protein mass spectrometry (MS) via electrospray-based methods is often degraded by low-mass contaminants, which can suppress the spectral quality of the analyte of interest via space-charge effects. Consequently, selective removal of contaminants by their mobilities would benefit native MS if achieved without additional hardware and before the mass analyzer regions used for selection, analyte readout, or tandem MS. Here, we use the high-pressure multipole within the source of an Orbitrap Tribrid as the foundation for a coarse ion filter. Using this method, we show complete filtration of 2 mM polyethylene glycol (PEG-1000) during native MS of SILu mAb antibody present at a 200× lower concentration. We also show the generality of the process by rescuing 10 µM tetrameric pyruvate kinase from 2 mM PEG-1000, asserting this voltage rollercoaster filtering (VRF) method for use in native MS as an efficient alternative to conventional purification methods.
Assuntos
Filtração/instrumentação , Polietilenoglicóis/isolamento & purificação , Proteínas/química , Animais , Anticorpos Monoclonais/química , Desenho de Equipamento , Humanos , Espectrometria de Massas/instrumentação , Piruvato Quinase/químicaRESUMO
Top-down proteomics studies intact proteoform mixtures and offers important advantages over more common bottom-up proteomics technologies, as it avoids the protein inference problem. However, achieving complete molecular characterization of investigated proteoforms using existing technologies remains a fundamental challenge for top-down proteomics. Here, we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. Notably, the described UVPD setup applies multiple laser pulses to induce ion dissociation, and this feature can be used to optimize the fragmentation outcome based on the molecular weight of the analyzed biomolecule. When applied to complex proteoform mixtures in high-throughput top-down proteomics, 213 nm UVPD demonstrated a high degree of complementarity with the most employed fragmentation method in proteomics studies, higher-energy collisional dissociation (HCD). UVPD at 213 nm offered higher average proteoform sequence coverage and degree of proteoform characterization (including localization of post-translational modifications) than HCD. However, previous studies have shown limitations in applying database search strategies developed for HCD fragmentation to UVPD spectra which contains up to nine fragment ion types. We therefore performed an analysis of the different UVPD product ion type frequencies. From these data, we developed an ad hoc fragment matching strategy and determined the influence of each possible ion type on search outcomes. By paring down the number of ion types considered in high-throughput UVPD searches from all types down to the four most abundant, we were ultimately able to achieve deeper proteome characterization with UVPD. Lastly, our detailed product ion analysis also revealed UVPD cleavage propensities and determined the presence of a product ion produced specifically by 213 nm photons. All together, these observations could be used to better elucidate UVPD dissociation mechanisms and improve the utility of the technique for proteomic applications.