RESUMO
Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling.
Assuntos
Sistema de Sinalização das MAP Quinases , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteômica/métodos , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Serina-Treonina Quinases TOR/metabolismo , Animais , Células Cultivadas , Marcação por Isótopo , Espectrometria de Massas , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Deleção de Sequência , Transdução de SinaisRESUMO
We have shown previously that coupling of high field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility, with liquid extraction surface analysis (LESA) mass spectrometry of tissue results in significant improvements in the resulting protein mass spectra. Here, we demonstrate LESA FAIMS mass spectrometry imaging of proteins in sections of mouse brain and liver tissue. The results are compared with LESA mass spectrometry images obtained in the absence of FAIMS. The results show that the number of different protein species detected can be significantly increased by incorporating FAIMS into the workflow. A total of 34 proteins were detected by LESA FAIMS mass spectrometry imaging of mouse brain, of which 26 were unique to FAIMS, compared with 15 proteins (7 unique) detected by LESA mass spectrometry imaging. A number of proteins were identified including α-globin, 6.8 kDa mitochondrial proteolipid, macrophage migration inhibitory factor, ubiquitin, ß-thymosin 4, and calmodulin. A total of 40 species were detected by LESA FAIMS mass spectrometry imaging of mouse liver, of which 29 were unique to FAIMS, compared with 24 proteins (13 unique) detected by LESA mass spectrometry imaging. The spatial distributions of proteins identified in both LESA mass spectrometry imaging and LESA FAIMS mass spectrometry imaging were in good agreement indicating that FAIMS is a suitable tool for inclusion in mass spectrometry imaging workflows.
Assuntos
Encéfalo/metabolismo , Fígado/metabolismo , Espectrometria de Massas/métodos , Proteínas/análise , Sequência de Aminoácidos , Animais , Encéfalo/patologia , Espectrometria de Mobilidade Iônica , Fígado/patologia , CamundongosRESUMO
AIM: Inflammatory periodontal disease is widespread in dogs. This study evaluated site-specific changes in the canine gingival crevicular fluid (GCF) proteome during longitudinal progression from very mild gingivitis to mild periodontitis. Periodontitis diagnosis in dogs requires general anaesthesia with associated risks and costs; our ultimate aim was to develop a periodontitis diagnostic for application in conscious dogs. The objective of this work was to identify potential biomarkers of periodontal disease progression in dogs. MATERIAL AND METHODS: Gingival crevicular fluid was sampled from a total of 10 teeth in eight dogs at three different stages of health/disease and samples prepared for quantitative mass spectrometry (data available via ProteomeXchange; identifier PXD003337). A univariate mixed model analysis determined significantly altered proteins between health states and six were evaluated by ELISA. RESULTS: Four hundred and six proteins were identified with 84 present in all samples. The prevalence of 40 proteins was found to be significantly changed in periodontitis relative to gingivitis. ELISA measurements confirmed that haptoglobin was significantly increased. CONCLUSIONS: This study demonstrates for the first time that proteins detected by mass spectrometry have potential to identify novel biomarkers for canine periodontal disease. Further work is required to validate additional biomarkers for a periodontitis diagnostic.
Assuntos
Gengivite , Periodontite , Animais , Modelos Animais de Doenças , Cães , Líquido do Sulco Gengival , Perda da Inserção Periodontal , ProteomaRESUMO
Glycosylation of flagellin is essential for the virulence of Campylobacter jejuni, a leading cause of bacterial gastroenteritis. Here, we demonstrate comprehensive mapping of the O-glycosylation of flagellin from Campylobacter jejuni 11168 by use of a bottom-up proteomics approach that incorporates differential ion mobility spectrometry (also known as high field asymmetric waveform ion mobility spectrometry or FAIMS) together with proteolysis with proteinase K. Proteinase K provides complementary sequence coverage to that achieved following trypsin proteolysis. The use of FAIMS increased the number of glycopeptides identified. Novel glycans for this strain were identified (pseudaminic acid and either acetamidino pseudaminic acid or legionaminic acid), as were novel glycosylation sites: Thr208, Ser343, Ser348, Ser349, Ser395, Ser398, Ser423, Ser433, Ser436, Ser445, Ser448, Ser451, Ser452, Ser454, Ser457 and Thr465. Multiply glycosylated peptides were observed, as well as variation at individual residues in the nature of the glycan and its presence or absence. Such extreme heterogeneity in the pattern of glycosylation has not been reported previously, and suggests a novel dimension in molecular variation within a bacterial population that may be significant in persistence of the organism in its natural environment. These results demonstrate the usefulness of differential ion mobility in proteomics investigations of PTMs.
Assuntos
Campylobacter jejuni/química , Flagelina/análise , Flagelina/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida , Endopeptidase K/metabolismo , Flagelina/metabolismo , Glicosilação , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Tripsina/metabolismoRESUMO
We have applied liquid chromatography high-field asymmetric waveform ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) to the investigation of site-specific phosphorylation in fibroblast growth factor (FGF) signaling. We have combined a SILAC approach with chemical inhibition by SU5402 (an FGF receptor tyrosine kinase inhibitor) and dasatinib (a Src family kinase inhibitor). The results show that incorporation of FAIMS within the workflow results in (a) an increase in the relative proportion of phosphothreonine and phosphotyrosine sites identified, (b) an increase in phosphopeptide identifications from precursors with charge states ≥ +3 (with an associated increase in peptide length), and (c) an increase in the identification of multiply phosphorylated peptides. Approximately 20% of the phosphorylation sites identified via the FAIMS workflow had not been reported previously, and over 80% of those were from multiply phosphorylated peptides. Moreover, FAIMS provided access to a distinct set of phosphorylation sites regulated in response to SU5402 and dasatinib. The enhanced identification of multiply phosphorylated peptides was particularly striking in the case of sites regulated by SU5402. In addition to providing a compelling example of the complementarity of FAIMS in phosphoproteomics, the results provide a valuable resource of phosphorylation sites for further investigation of FGF signaling and trafficking.
Assuntos
Cromatografia Líquida/métodos , Fatores de Crescimento de Fibroblastos/metabolismo , Espectrometria de Massas/métodos , Fosfopeptídeos/análise , Fosfopeptídeos/metabolismo , Sequência de Aminoácidos , Dasatinibe/farmacologia , Humanos , Dados de Sequência Molecular , Fosforilação , Fosfotirosina/análise , Fosfotirosina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Pirróis/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Espectrometria de Massas em Tandem/métodos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Previously we have shown that liquid extraction surface analysis (LESA) mass spectrometry is suitable for the analysis of intact proteins from a range of biological substrates. Here we show that LESA mass spectrometry may be coupled with high field asymmetric waveform ion mobility spectrometry (FAIMS) for top-down protein analysis directly from thin tissue sections (mouse liver, mouse brain) and from bacterial colonies (Escherichia coli) growing on agar. Incorporation of FAIMS results in significant improvements in signal-to-noise and reduced analysis time. Abundant protein signals are observed in single scan mass spectra. In addition, FAIMS enables gas-phase separation of molecular classes, for example, lipids and proteins, enabling improved analysis of both sets of species from a single LESA extraction.
Assuntos
Espectrometria de Massas/métodos , Proteínas/análise , Animais , Camundongos , Propriedades de SuperfícieRESUMO
Liquid extraction surface analysis (LESA) is a surface sampling technique that allows electrospray mass spectrometry analysis of a wide range of analytes directly from biological substrates. Here, we present LESA mass spectrometry coupled with high field asymmetric waveform ion mobility spectrometry (FAIMS) for the analysis of dried blood spots on filter paper. Incorporation of FAIMS in the workflow enables gas-phase separation of lipid and protein molecular classes, enabling analysis of both haemoglobin and a range of lipids (phosphatidylcholine or phosphatidylethanolamine, and sphingomyelin species) from a single extraction sample. The work has implications for multiplexed clinical assays of multiple analytes.
Assuntos
Teste em Amostras de Sangue Seco/métodos , Extração Líquido-Líquido/métodos , Espectrometria de Massas/métodos , Adulto , Hemoglobinas/análise , Hemoglobinas/isolamento & purificação , Humanos , Lipídeos/sangue , Lipídeos/isolamento & purificação , Propriedades de SuperfícieRESUMO
Large scale analysis of proteins by mass spectrometry is becoming increasingly routine; however, the presence of peptide isomers remains a significant challenge for both identification and quantitation in proteomics. Classes of isomers include sequence inversions, structural isomers, and localization variants. In many cases, liquid chromatography is inadequate for separation of peptide isomers. The resulting tandem mass spectra are composite, containing fragments from multiple precursor ions. The benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) for proteomics have been demonstrated by a number of groups, but previously work has focused on extending proteome coverage generally. Here, we present a systematic study of the benefits of FAIMS for a key challenge in proteomics, that of peptide isomers. We have applied FAIMS to the analysis of a phosphopeptide library comprising the sequences GPSGXVpSXAQLX(K/R) and SXPFKXpSPLXFG(K/R), where X = ADEFGLSTVY. The library has defined limits enabling us to make valid conclusions regarding FAIMS performance. The library contains numerous sequence inversions and structural isomers. In addition, there are large numbers of theoretical localization variants, allowing false localization rates to be determined. The FAIMS approach is compared with reversed-phase liquid chromatography and strong cation exchange chromatography. The FAIMS approach identified 35% of the peptide library, whereas LC-MS/MS alone identified 8% and LC-MS/MS with strong cation exchange chromatography prefractionation identified 17.3% of the library.
Assuntos
Fosfopeptídeos/química , Proteômica/métodos , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia de Fase Reversa , Bases de Dados de Proteínas , Isomerismo , Dados de Sequência Molecular , Biblioteca de Peptídeos , Fosforilação , Espectrometria de Massas em TandemRESUMO
Large scale mass spectrometry analysis of N-linked glycopeptides is complicated by the inherent complexity of the glycan structures. Here, we evaluate a mass spectrometry approach for the targeted analysis of N-linked glycopeptides in complex mixtures that does not require prior knowledge of the glycan structures or pre-enrichment of the glycopeptides. Despite the complexity of N-glycans, the core of the glycan remains constant, comprising two N-acetylglucosamine and three mannose units. Collision-induced dissociation (CID) mass spectrometry of N-glycopeptides results in the formation of the N-acetylglucosamine (GlcNAc) oxonium ion and a [mannose+GlcNAc] fragment (in addition to other fragments resulting from cleavage within the glycan). In ion-trap CID, those ions are not detected due to the low m/z cutoff; however, they are detected following the beam-type CID known as higher energy collision dissociation (HCD) on the orbitrap mass spectrometer. The presence of these product ions following HCD can be used as triggers for subsequent electron transfer dissociation (ETD) mass spectrometry analysis of the precursor ion. The ETD mass spectrum provides peptide sequence information, which is unobtainable from HCD. A Lys-C digest of ribonuclease B and trypsin digest of immunoglobulin G were separated by ZIC-HILIC liquid chromatography and analyzed by HCD product ion-triggered ETD. The data were analyzed both manually and by search against protein databases by commonly used algorithms. The results show that the product ion-triggered approach shows promise for the field of glycoproteomics and highlight the requirement for more sophisticated data mining tools.
Assuntos
Glicoproteínas/análise , Glicoproteínas/química , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Animais , Bovinos , Glicosilação , Humanos , Íons/análise , Íons/química , Dados de Sequência Molecular , Processamento de Proteína Pós-TraducionalRESUMO
The analysis of intact glycopeptides by mass spectrometry is challenging due to the numerous possibilities for isomerization, both within the attached glycan and the location of the modification on the peptide backbone. Here, we demonstrate that high field asymmetric wave ion mobility spectrometry (FAIMS), also known as differential ion mobility, is able to separate isomeric O-linked glycopeptides that have identical sequences but differing sites of glycosylation. Two glycopeptides from the glycoprotein mucin 5AC, GT(GalNAc)TPSPVPTTSTTSAP and GTTPSPVPTTST(GalNAc)TSAP (where GalNAc is O-linked N-acetylgalactosamine), were shown to coelute following reversed-phase liquid chromatography. However, FAIMS analysis of the glycopeptides revealed that the compensation voltage ranges in which the peptides were transmitted differed. Thus, it is possible at certain compensation voltages to completely separate the glycopeptides. Separation of the glycopeptides was confirmed by unique reporter ions produced by supplemental activation electron transfer dissociation mass spectrometry. These fragments also enable localization of the site of glycosylation. The results suggest that glycan position plays a key role in determining gas-phase glycopeptide structure and have implications for the application of FAIMS in glycoproteomics.
Assuntos
Glicopeptídeos/análise , Íons/química , Espectrometria de Massas por Ionização por Electrospray , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Glicopeptídeos/isolamento & purificação , Glicosilação , Isomerismo , Dados de Sequência Molecular , EspectrofotometriaRESUMO
Alzheimer's disease (AD) is a devastating neurodegenerative disorder that has been predicted to affect 106.2 million people worldwide by 2050. Currently, definitive diagnosis for this disease is given post mortem, and there is a need for biomarker identification to enable earlier diagnosis of this disease. Biomarkers of AD would ideally represent early disease process and will be present in peripheral tissue before cognitive decline develops in this population. Proteomic technologies offer a strategy to undertake such work. In recent times, research in this field has moved away from classical 2-dimensional gel-based proteomics toward more sensitive, non-gel-based proteomic methodologies. In the study presented here, isobaric labeling for relative and absolute quantification was used to assess plasma protein expression in a small group of AD and control samples. Several proteins were identified as being differentially expressed between these 2 populations. Complement 4a plasma protein was identified as increased in AD by isobaric labeling for relative and absolute quantification, and this finding was further validated by Western blotting and enzyme-linked immunosorbent assay. These data suggest that inflammatory processes, which have been shown to be involved in AD pathology in the brain, are also present in plasma.
Assuntos
Doença de Alzheimer/sangue , Biomarcadores/sangue , Complemento C4a/análise , Idoso , Western Blotting , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Humanos , Espectrometria de Massas , Proteômica/métodosRESUMO
Hemoglobinopathies are the most common inherited disorders. Newborn blood screening for clinically significant hemoglobin variants, including sickle (HbS), HbC, and HbD, has been adopted in many countries as it is widely acknowledged that early detection improves the outcome. We present a method for determination of Hb variants by direct surface sampling of dried blood spots by use of an Advion Triversa Nanomate automated electrospray system coupled to a high-resolution mass spectrometer. The method involves no sample preparation. It is possible to unambiguously identify homozygous and heterozygous HbS, HbC, and HbD variants in <10 min without the need for additional confirmation. The method allows for repeated analysis of a single blood spot over a prolonged time period and is tolerant of blood spot storage conditions.
Assuntos
Análise Química do Sangue/métodos , Coleta de Amostras Sanguíneas/métodos , Hemoglobinas Anormais/análise , Espectrometria de Massas/métodos , Adulto , Hemoglobinas Anormais/isolamento & purificação , Humanos , Recém-Nascido , Fatores de TempoRESUMO
Protein identification in bottom-up proteomics requires disentangling isomers of proteolytic peptides, a major class of which are sequence inversions. Their separation using ion mobility spectrometry (IMS) has been limited to isomeric pairs. Here we demonstrate baseline separation of all seven 8-mer tryptic peptide isomers using differential IMS. Evaluation of peak capacity implies that even larger libraries should be resolved for heavier peptides with higher charge states.
Assuntos
Espectrometria de Massas/métodos , Peptídeos/química , Sequência de Aminoácidos , Íons/química , Isomerismo , Peptídeos/isolamento & purificação , Proteômica , Tripsina/metabolismoRESUMO
The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC-MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.
Assuntos
Gengivite/metabolismo , Inflamação/metabolismo , Proteoma/química , Proteômica/métodos , Doença Aguda , Adulto , Cromatografia Líquida , Análise por Conglomerados , Feminino , Fusobacterium/química , Líquido do Sulco Gengival/química , Gengivite/microbiologia , Humanos , Inflamação/microbiologia , Marcação por Isótopo , Masculino , Metagenoma , Modelos Biológicos , Proteoma/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Estatísticas não Paramétricas , Espectrometria de Massas em TandemRESUMO
Many proteins and proteolytic peptides incorporate the same post-translational modification (PTM) at different sites, creating multiple localization variants with different functions or activities that may coexist in cells. Current analytical methods based on liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS) are challenged by such isomers that often coelute in LC and/or produce nonunique fragment ions. The application of ion mobility spectrometry (IMS) was explored, but success has been limited by insufficient resolution. We show that high-resolution differential ion mobility spectrometry (FAIMS) employing helium-rich gases can readily separate phosphopeptides with variant modification sites. Use of He/N(2) mixtures containing up to 74% He has allowed separating to >95% three monophosphorylated peptides of identical sequence. Similar separation was achieved at 50% He, using an elevated electric field. Bisphosphorylated isomers that differ in only one modification site were separated to the same extent. We anticipate FAIMS capabilities for such separations to extend to other PTMs.
Assuntos
Fosfopeptídeos/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Isomerismo , Dados de Sequência Molecular , Fosfopeptídeos/isolamento & purificaçãoRESUMO
Fibroblast Growth Factor (FGF) dependent signalling is frequently activated in cancer by a variety of different mechanisms. However, the downstream signal transduction pathways involved are poorly characterised. Here a quantitative differential phosphoproteomics approach, SILAC, is applied to identify FGF-regulated phosphorylation events in two triple- negative breast tumour cell lines, MFM223 and SUM52, that exhibit amplified expression of FGF receptor 2 (FGFR2) and are dependent on continued FGFR2 signalling for cell viability. Comparative Gene Ontology proteome analysis revealed that SUM52 cells were enriched in proteins associated with cell metabolism and MFM223 cells enriched in proteins associated with cell adhesion and migration. FGFR2 inhibition by SU5402 impacts a significant fraction of the observed phosphoproteome of these cells. This study expands the known landscape of FGF signalling and identifies many new targets for functional investigation. FGF signalling pathways are found to be flexible in architecture as both shared, and divergent, responses to inhibition of FGFR2 kinase activity in the canonical RAF/MAPK/ERK/RSK and PI3K/AKT/PDK/mTOR/S6K pathways are identified. Inhibition of phosphorylation-dependent negative-feedback pathways is observed, defining mechanisms of intrinsic resistance to FGFR2 inhibition. These findings have implications for the therapeutic application of FGFR inhibitors as they identify both common and divergent responses in cells harbouring the same genetic lesion and pathways of drug resistance.
Assuntos
Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Ontologia Genética , Humanos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidoresRESUMO
Large data sets of electron capture dissociation (ECD) mass spectra from proteomic experiments are rich in information; however, extracting that information in an optimal manner is not straightforward. Protein database search engines currently available are designed for low resolution CID data, from which Fourier transform ion cyclotron resonance (FT-ICR) ECD data differs significantly. ECD mass spectra contain both z-prime and z-dot fragment ions (and c-prime and c-dot); ECD mass spectra contain abundant peaks derived from neutral losses from charge-reduced precursor ions; FT-ICR ECD spectra are acquired with a larger precursor m/z isolation window than their low-resolution CID counterparts. Here, we consider three distinct stages of postacquisition analysis: (1) processing of ECD mass spectra prior to the database search; (2) the database search step itself and (3) postsearch processing of results. We demonstrate that each of these steps has an effect on the number of peptides identified, with the postsearch processing of results having the largest effect. We compare two commonly used search engines: Mascot and OMSSA. Using an ECD data set of modest size (3341 mass spectra) from a complex sample (mouse whole cell lysate), we demonstrate that search results can be improved from 630 identifications (19% identification success rate) to 1643 identifications (49% identification success rate). We focus in particular on improving identification rates for doubly charged precursors, which are typically low for ECD fragmentation. We compare our presearch processing algorithm with a similar algorithm recently developed for electron transfer dissociation (ETD) data.
Assuntos
Bases de Dados de Proteínas , Espectrometria de Massas/métodos , Proteômica/métodos , Animais , Células/química , Espectrometria de Massas/instrumentação , Espectrometria de Massas/normas , Camundongos , Ferramenta de Busca/normasRESUMO
We have applied high-field asymmetric waveform ion mobility spectrometry (FAIMS) to the analysis of the phosphopeptides APLpSFRGSLPKSYVK, APLSFRGpSLPKSYVK, and APLSFRGSLPKpSYVK. The peptides have identical amino acid sequences and differ only in the site of phosphorylation. The results show that FAIMS is capable of at least partially separating these species. Separation was confirmed by coupling FAIMS with high-resolution electron transfer dissociation (ETD) mass spectrometry. Phosphorylation is retained on the ETD peptide fragments thereby allowing assignment of the site of the modification. Co-eluting phosphopeptides which differ only in the site of modification are frequently observed in liquid chromatography/tandem mass spectrometry phosphoproteomics experiments, and therefore these proof-of-principle results have implications for the application of FAIMS in that field.
Assuntos
Espectrometria de Massas/métodos , Fosfopeptídeos/química , Espectrometria de Massas/instrumentação , Fosfopeptídeos/síntese químicaRESUMO
The effect of site and frequency of phosphorylation on the electron capture dissociation of peptide ions has been investigated. The ECD of a suite of synthetic peptides (APLSFRGSLPKSYVK; one unmodified, three singly-phosphorylated, three-doubly phosphorylated, and one triply-phosphorylated); two tryptic phosphopeptides (YKVPQLEIVPN(p)SAEER, alpha-casein and FQ(p)SEEQQQTEDELQDK, beta-casein) and their unmodified counterparts, were determined over a range of ECD cathode potentials. The results show that, for doubly-charged precursor ions, the presence of phosphorylation has a deleterious effect on ECD sequence coverage. The fragmentation patterns observed suggest that for peptides with multiple basic residues, the phospho-groups exist in their deprotonated form and form salt-bridges with protonated amino acid side chains. The fragmentation observed for the acidic tryptic peptides suggested the presence of noncovalent interactions, which were perturbed on phosphorylation. Increasing the ECD electron energy significantly improves sequence coverage. Alternatively, improved sequence coverage can be achieved by performing ECD on triply-charged precursor ions. The findings are important for the understanding of gas-phase fragmentation of phosphopeptides.
Assuntos
Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Animais , Caseínas/química , Bovinos , Elétrons , Íons , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosforilação , Tripsina/químicaRESUMO
Electron capture dissociation (ECD) offers many advantages over the more traditional fragmentation techniques for the analysis of peptides and proteins, although the question remains: How suitable is ECD for incorporation within proteomic strategies for the identification of proteins? Here, we compare LC-ECD-MS/MS and LC-CID-MS/MS as techniques for the identification of proteins. Experiments were performed on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin, lysozyme, cytochrome c, alcohol dehydrogenase, and beta-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides. The results show that although protein coverage was lower for LC-ECD-MS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags, providing greater confidence in protein assignment.