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1.
Glycoconj J ; 38(2): 213-231, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33835347

RESUMO

Pancreatic cancer is a highly malignant tumor of the digestive tract that is difficult to diagnose and treat. It is more common in developed countries and has become one of the main causes of death in some countries and regions. Currently, pancreatic cancer generally has a poor prognosis, partly due to the lack of symptoms in the early stages of pancreatic cancer. Therefore, most cases are diagnosed at advanced stage. With the continuous in-depth research of glycoproteomics in precision medical diagnosis, there have been some reports on quantitative analysis of cancer-related cells, plasma or tissues to find specific biomarkers for targeted therapy. This research is based on the developed complete N-linked glycopeptide database search engine GPSeeker, combined with liquid-mass spectrometry and stable diethyl isotope labeling, providing a benchmark of site- and structure-specific quantitative tissue N-glycoproteomics for discovery of potential N-glycoprotein markers. With spectrum-level FDR ≤1%, 20,038 intact N-Glycopeptides corresponding to 4518 peptide backbones, 228 N-glycan monosaccharide compositions 1026 N-glycan putative structures, 4460 N-glycosites and 3437 intact N-glycoproteins were identified. With the criteria of ≥1.5-fold change and p value<0.05, 52 differentially expressed intact N-glycopeptides (DEGPs) were found in pancreatic cancer tussues relative to control, where 38 up-regulated and 14 down-regulated, respectively.


Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Neoplasias Pancreáticas/metabolismo , Benchmarking , Biomarcadores Tumorais/metabolismo , Bases de Dados de Proteínas , Glicoproteínas/análise , Humanos , Marcação por Isótopo , Proteômica/métodos , Ferramenta de Busca , Espectrometria de Massas em Tandem/métodos
2.
Rapid Commun Mass Spectrom ; 35(1): e8952, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32965048

RESUMO

RATIONALE: N-glycosylation is one of the most common protein post-translational modifications; it is extremely complex with multiple glycoforms from different monosaccharide compositions, sequences, glycosidic linkages, and anomeric positions. Each glycoform functions with a particular site- and structure-specific N-glycan that can be fully characterized using state-of-the-art tandem mass spectrometry (MS/MS) and the intact N-glycopeptide database search engine GPSeeker that we recently developed. Urine has recently gained increasing attention as a non-invasive source for disease marker discovery. In this study, we report our structure-specific N-glycoproteomics study of human urine. METHODS: We performed trypsin digestion, Zwitterionic Hydrophilic Interaction chromatography (ZIC-HILIC) enrichment, C18-RPLC/nano-ESI-MS/MS using HCD with stepped normalized collisional energies, and GPSeeker database search for a comprehensive site- and structure-specific N-glycoproteomics characterization of the human urinary N-glycoproteome at the intact N-glycopeptide level. For this, we used b/y product ion pairs from the GlcNAc-containing site-determining peptide backbone and structure-diagnostic product ions from the N-glycan moieties, respectively. RESULTS: We identified 2986 intact N-glycopeptides with comprehensive site and structure information for the peptide backbones (amino acid sequences and N-glycosites) and the N-glycan moieties (monosaccharide compositions, sequences/linkages). The 2986 intact N-glycopeptide IDs corresponded to 754 putative N-glycan linkage structures on 419 N-glycosites of 450 peptide backbones from 327 intact N-glycoproteins. Next, 146 linkage structures and 200 N-glycosites were confirmed with structure-diagnostic and GlcNAc-containing site-determining product ions, respectively. CONCLUSIONS: We found 106 new N-glycosites not annotated in the current UniProt database. The elution-abundance patterns of urinary intact N-glycopeptide oxonium ions (m/z 138 and 204) of the same subject were temporally stable during the day and over 6 months. These patterns are rather different among different subjects. The results implied an interesting possibility that glycopeptide oxonium ion patterns could serve as distinguishing markers between individuals and/or between physiological and pathological states.


Assuntos
Glicopeptídeos , Glicoproteínas , Proteômica/métodos , Sequência de Carboidratos , Cromatografia de Fase Reversa/métodos , Glicopeptídeos/química , Glicopeptídeos/urina , Glicoproteínas/química , Glicoproteínas/urina , Glicosilação , Humanos , Íons/química , Íons/urina , Masculino , Proteoma/análise , Espectrometria de Massas em Tandem/métodos
3.
Phenomics ; 1(6): 269-284, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36939756

RESUMO

Currently, drug resistance of anti-cancer therapy has become the main cause of low survival rate and poor prognosis. Full understanding of drug resistance mechanisms is an urgent request for further development of anti-cancer therapy and improvement of prognosis. Here we present our N-glycoproteomics study of putative N-glycoprotein biomarkers of drug resistance in doxorubicin resistance breast cancer cell line michigan cancer foundation-7 (MCF-7/ADR) relative to parental michigan cancer foundation-7 (MCF-7) cells. Intact N-glycopeptides (IDs) from MCF-7/ADR and MCF-7 cells were enriched with zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), labeled with stable isotopic diethylation (SIDE), and analyzed with C18-RPLC-MS/MS (HCD with stepped normalized collision energies); these IDs were identified with database search engine GPSeeker, and the differentially expressed intact N-glycopeptides (DEGPs) were quantified with GPSeekerQuan. With target-decoy searches and control of spectrum-level FDR ≤ 1%, 322 intact N-glycopeptides were identified; these intact N-glycopeptides come from the combination of 249 unique peptide backbones (corresponding to 234 intact N-glycoproteins) and 90 monosaccharide compositions (corresponding to 248 putative N-glycosites). The sequence structures of 165 IDs were confirmed with structure-diagnostic fragment ions. With the criteria of observation at least twice among the three technical replicates, ≥ 1.5-fold change and p value < 0.05, 20 DEGPs were quantified, where five of them were up-regulated and 15 of them were down-regulated; the corresponding intact N-glycoproteins as putative markers of drug resistance were discussed. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-021-00029-8.

4.
Talanta ; 219: 121359, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32887082

RESUMO

Protein N-glycosylation plays an essential role on cancers and other pathological processes. Its structural and functional studies rely on complete qualitative and quantitative information. Thus, it is important to quantify differentially expressed intact N-glycopeptides (DEGPs) at their molecular level. Here we report our application of stable isotopic diethyl labeling (SIDE) of amino groups for relative quantitation of intact N-glycopeptides. The amino groups from both the N-terminal and lysine residues of N-glycopeptides were diethylated with XH3XHO (X = 13C or C) and NaBH3CN. A linear quantitation dynamic range up to 50-fold was obtained with R2 = 0.9985 with intact N-glycopeptides from standard glycoprotein ribonuclease B (RNase B). In proof-of-principle comparative N-glycoproteomics study of aberrant N-glycosylation of gastric cancer tissues vs. adjacent tissues using SIDE, 644 DEGPs (≥1.5-fold change, p < 0.05, p was calculated using t-test) were discovered. With its accuracy and big dynamic range, SIDE can be applied to quantitative study of any aberrant glycosylation at the intact glycopeptide level.


Assuntos
Proteômica , Espectrometria de Massas em Tandem , Glicopeptídeos , Glicosilação , Marcação por Isótopo
5.
Clin Proteomics ; 17: 3, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32042278

RESUMO

BACKGROUND: Cancer stem cells (CSCs) are reported to be responsible for tumor initiation, progression, metastasis, and therapy resistance where P-glycoprotein (P-gp) as well as other glycoproteins are involved. Identification of these glycoprotein markers is critical for understanding the resistance mechanism and developing therapeutics. METHODS: In this study, we report our comparative and quantitative site- and structure-specific N-glycoproteomics study of MCF-7/ADR cancer stem cells (CSCs) vs. MCF-7/ADR cells. With zic-HILIC enrichment, isotopic diethyl labeling, RPLC-MS/MS (HCD) analysis and GPSeeker DB search, differentially expressed N-glycosylation was quantitatively characterized at the intact N-glycopeptide level. RESULTS: 4016 intact N-glycopeptides were identified with spectrum-level FDR ≤ 1%. With the criteria of ≥ 1.5 fold change and p value < 0.05, 247 intact N-glycopeptides were found differentially expressed in MCF-7/ADR CSCs as putative markers. Raw data are available via ProteomeXchange with identifier PXD013836. CONCLUSIONS: Quantitative site- and structure-specific N-glycoproteomics characterization may help illustrate the cell stemness property.

6.
J Proteomics ; 212: 103594, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31759178

RESUMO

Glycosylation is a common protein PTM, and its aberrant regulation has been widely linked to various pathological conditions including cancers. Our recent development of intact N-glycopeptide search engine GPSeeker has enabled relatively quantitative structure-specific characterization of differentially expressed intact N-glycopeptides (DEGPs) with isotopic labeling of the peptide backbones and structure-specific fragment ions of the N-glycan moieties. Here we report our site- and structure-specific relatively quantitative N-glycoproteomics study of breast MCF-7 cancer cells (relative to epithelial MCF-10A cells) using RPLC-nanoESI-MS/MS and GPSeeker. With spectrum-level FDR ≤ 1%, 581 intact N-glycopeptides with comprehensive structural information of both the peptide backbones (amino acid sequences, N-glycosites) and the N-glycan moieties (monosaccharide composition, sequence and linkages) were identified from five technical replicates (TR1, TR2, TR3, TR4 and TR5). With the criteria of quantified at least thrice out of the five technical replicates with no <1.5-fold change, p ≤ .05 and RSD ≤ 20%, 56 DEGPs were quantified from 23 N-glycosites on 19 intact N-glycoproteins. For the 19 intact N-glycoproteins observed with differential N-glycosylation expression, 14 (each with one or more DEGPs) were observed with uniform down regulation; 6 (each with one or more DEGPs) were observed with uniform up regulation; whereas one was observed with both up and down regulation. SIGNIFICANCE: Differential N-glycosylation in breast MCF-7 cancer cells (relative to MCF-10A epithelial cells) were qualitatively and quantitatively characterized with site- and structure-specific N-glycoproteomics using RPLC-nanoESI-MS/MS (HCD with stepped NCEs) and intact N-glycopeptide search engine GPSeeker. With spectrum-level FDR ≤ 1%, 581 intact N-glycopeptides with comprehensive structural information of both the peptide backbones and the N-glycan moieties were identified; For the 248 putative N-glycosites, 248 were confirmed where 125 have not been annotated in UniProt as of July 25, 2019. For the 114 N-glycan putative linkage structures, 44 were confirmed with no less than one structure-diagnostic fragment ions. With the criteria of quantified at least thrice out of the five technical replicates with no < 1.5-fold change and p ≤ .05, 56 DEGPs were quantified from 21 intact N-glycoproteins; 13 and 5 intact N-glycoproteins (each with one or more DEGPs) were observed with uniform down and up regulation; whereas one were observed with simultaneous up and down regulation.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Glicopeptídeos/análise , Glicoproteínas/análise , Polissacarídeos/análise , Proteômica/métodos , Sequência de Aminoácidos , Linhagem Celular , Biologia Computacional , Feminino , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Humanos , Células MCF-7 , Polissacarídeos/química , Ferramenta de Busca , Software , Espectrometria de Massas em Tandem/métodos
7.
Rapid Commun Mass Spectrom ; 34(6): e8622, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31658499

RESUMO

RATIONALE: We previously isolated antibodies binding to glycopeptide neoantigen epitopes centering the GSTA sequence of the highly glycosylated tandem repeat region of MUC1. Epitopes centering the GSTA sequence are also predicted by NetMHC programs to bind to MHC molecules. Detecting MUC1 glycopeptide epitopes remains a challenge since antigenic epitopes are often shorter than 10 amino acids. METHODS: In this study, we used pronase from Streptomyces griseus, which has no amino acid sequence preference for enzymatic cleavage sites, to digest synthetic glycopeptides RPAPGST (Tn)APPAHG and RPAPGS (Tn)TAPPAHG, and analyzed the digests by liquid chromatography/mass spectrometry (LC/MS) using electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) methods with an Orbitrap Fusion Lumos Tribid mass spectrometer. RESULTS: We found that short glycopeptides containing 8 to 11 amino acids could be efficiently generated by pronase digestion. Such glycopeptides of minimal epitope lengths were clearly distinguished by characteristic MS/MS ion patterns and LC elution profiles. A glycopeptide library was generated which may serve as a standard for measuring neoantigen epitopes centering the GSTA sequence. CONCLUSIONS: Our data established the LC/MS/MS identities of a clinically relevant MUC1 glycopeptide neoantigen epitope centering the GSTA motif. A library of short MUC1 glycopeptides centered on the GSTA motif was created, which is a critical step for analysis of such antigen epitopes in real biological samples.


Assuntos
Epitopos/análise , Glicopeptídeos/análise , Mucina-1/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Cromatografia Líquida , Glicosilação , Humanos , Espectrometria de Massas em Tandem
8.
Anal Chim Acta ; 1091: 1-22, 2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31679562

RESUMO

N-glycosylation is one of the most frequently occurring protein post-translational modifications (PTMs) with broad cellular, physiological and pathological relevance. Mass spectrometry-based N-glycomics has become the state-of-the-art instrumental analytical pipeline for sensitive, high-throughput and comprehensive characterization of N-glycans and N-glycomes. Improvement and new development of methods in N-glycan release, enrichment, derivatization, isotopic labeling, separation, ionization, MS, tandem MS and informatics accompany side-by-side wider and deeper application. This review provides a comprehensive update of mass spectrometry-based qualitative and quantitative N-glycomics in the years of 2017-2018.


Assuntos
Glicômica/métodos , Polissacarídeos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Sequência de Carboidratos , Linhagem Celular , Humanos , Vírus da Influenza A Subtipo H9N2/química
9.
Curr Protoc Protein Sci ; 97(1): e94, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31517451

RESUMO

Site- and structure-specific quantitative N-glycoproteomics characterization of differentially expressed N-glycosylation at the intact N-glycopeptide level with distinct chromatographic separation and structure-specific fragment ions has become possible with the recent development of RPLC-pentaHILIC 2DLC separation and use of the intact N-glycopeptide search engine GPSeeker. Here we provide a detailed protocol for this GPSeeker-centered structure-specific isotopic-labeling quantitative N-glycoproteomics pipeline. The protocols include sample preparation of a 1:1 mixture of light (-CH3 )2 and heavy (-13 CD2 H)2 dimethylated intact N-glycopeptides from LO2 and HepG2 cells, RPLC-pentaHILIC 2DLC separation of the mixture, intact N-glycopeptide database search and identification using GPSeeker, and quantitation of differentially expressed intact N-glycopeptides using the quantitation module GPSeekerQuan. © 2019 by John Wiley & Sons, Inc.


Assuntos
Extratos Celulares/análise , Extratos Celulares/isolamento & purificação , Glicopeptídeos/análise , Glicopeptídeos/isolamento & purificação , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Glicosilação , Conformação Proteica , Proteômica/métodos , Ferramenta de Busca/métodos , Espectrometria de Massas em Tandem
10.
Chem Commun (Camb) ; 55(55): 7934-7937, 2019 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-31219116

RESUMO

High-throughput proteome-level characterization of stemness markers of MCF-7 cancer stem cells was carried out using our recently developed site- and structure-specific isotopic-labeling quantitative N-glycoproteomics pipeline. 144 differentially expressed intact N-glycopeptides were quantified all at once yet with good accurate consistency with the individual biochemical measurements of proteins, RNA or DNA.


Assuntos
Glicoproteínas/análise , Proteoma/análise , Sequência de Aminoácidos , Biomarcadores/análise , Regulação para Baixo , Ontologia Genética , Humanos , Marcação por Isótopo , Células MCF-7 , Células-Tronco Neoplásicas/química , Proteômica/métodos , Regulação para Cima
11.
J Am Soc Mass Spectrom ; 30(7): 1254-1261, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31098956

RESUMO

N-linked glycosylation is one of the most common protein PTMs, and the topological structure (monosaccharide composition and sequence as well as glycosidic linkages) of N-glycans is vital information to understand their biological functions and roles. Tandem mass spectrometry has been widely used for topological structure characterization of N-glycans, where comprehensive understanding of fragmentation pathways and characteristics of product ions are essential to achieve best interpretation of MS/MS data and highest confidence of identification. Here, we report our glycomic study of N-glycome of mouse brain as well as fragmentation pathway analysis of the identified N-glycans. With LC-MS/MS analysis at both the positive and negative ESI modes together with our recently developed N-glycan database search engine GlySeeker, 221 unique N-glycans with putative topological structures were identified with target-decoy searches and number of best hits of 1. Analysis of fragmentation pathways and characteristics of product ions of permethylated N-glycans in the positive mode and native N-glycans in the negative mode were further carried out. The reported N-glycans serve as a basic reference for future glycosylation study of mouse brain; and in general database search of tandem mass spectra of N-glycans, B/Y/Z ions should be preferentially considered for the permethylated form in the positive mode and B/C/Z ions for the native form in the negative mode.


Assuntos
Química Encefálica , Polissacarídeos/análise , Animais , Feminino , Glicômica , Camundongos Endogâmicos C57BL , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
12.
J Proteome Res ; 18(7): 2885-2895, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31117584

RESUMO

N-Glycosylation, being one of the most common and complex protein post-translational modifications (PTMs), is known to have microheterogeneity with the presence of different N-glycan structures at a single specific glycosite. These different structures may have exactly the same monosaccharide composition but totally different differential expressions and pathological relevance. Mass spectrometry-based N-glycoproteomics has so far been successful in large-scale characterization of these N-glycans at the composition level, and structure-level identification and quantitation is urgently needed. Here we report our development of the intact N-glycopeptide search engine GPSeeker and the GPSeeker-centered quantitative structural N-glycoproteomics pipeline. In benchmark characterization of differentially expressed N-glycosylation in hepatocellular carcinoma HepG2 cells relative to LO2 cells, 5 405 and 1 081 intact N-glycopeptides with putative linkage structures were identified and quantified with isotopic dimethyl labeling and 2D liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Among the 5 405 IDs, 837 were identified with no less than one structure diagnostic fragment ion for the N-glycan moieties. Besides double isomers of sialic acid linkages and fucose sequences, quadruple isomers from combination of two linages and two sequences were chromatographically separated and confidently identified; microheterogeneity with different differentially expressions were observed on 183 out of the 231 quantified N-glycosites. This GPSeeker-centered quantitative structural N-glycoproteomics pipeline can be widely applied to precise qualitative and quantitative characterization of N-glycosylation with physiological and pathological relevance.


Assuntos
Carcinoma Hepatocelular/química , Glicopeptídeos/análise , Proteômica/métodos , Ferramenta de Busca , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Glicosilação , Células Hep G2 , Humanos , Polissacarídeos/análise , Espectrometria de Massas em Tandem/métodos
13.
J Mass Spectrom ; 54(3): 258-265, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30698319

RESUMO

Histone post-translational modifications (PTMs) play various roles in chromatin-related cellular processes, and comprehensive analysis of these combinatorial PTMs at the intact protein level by top-down proteomics is the method of choice to reveal their crosstalk and biological functions. Here, we report our top-down characterization of the core histones from mouse fibroblasts cells NIH/3T3, which is a classic model used in many kinds of research. With nanoRPLC-MS/MS analysis and ProteinGoggle database search, 547 protein species were identified with spectrum-level FDR ≤ 1%, where PTMs in 51 protein species were unambiguously localized with PTM scores ≥1. High-resolution MS/MS data also allowed the unambiguous identification of acetylation instead of trimethylation. This study presents a general picture of combinatorial PTMs of mouse core histones, which serves as a basic reference for all future related biological studies.


Assuntos
Histonas/análise , Células 3T3 , Acetilação , Animais , Bases de Dados de Proteínas , Metilação , Camundongos , Processamento de Proteína Pós-Traducional , Proteômica
14.
J Proteome Res ; 18(1): 372-379, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30343578

RESUMO

Cell-surface N-glycans play important roles in both inter- and intracellular processes, including cell adhesion and development, cell recognition, as well as cancer development and metastasis; detailed structural characterization of these N-glycans is thus paramount. Here we report our comparative N-glycomics study of cell-surface N-glycans of the hepatocellular carcinoma (HCC) HepG2 cells vs the normal liver LO2 cells. With sequential trypsin digestion of proteins, C18 depletion of peptides without glycosylation, PNGase F digestion of N-glycopeptides, PGC enrichment of N-glycans, CH3I permethylation of the enriched N-glycans, cell-surface N-glycomes of the HepG2 and LO2 cells were analyzed using C18-RPLC-MS/MS (HCD). With spectrum-level FDR no bigger than 1%, 351 and 310 N-glycans were identified for HepG2 and LO2, respectively, with comprehensive structural information (not only monosaccharide composition, but also sequence and linkage) by N-glycan database search engine GlySeeker. The percentage of hybrid N-glycans with tetra-antennary structures was substantially increased in the HepG2 cells. This comprehensive discovery study of differentially expressed cell-surface N-glycans in HepG2 vs LO2 serves as a solid reference for future validation study of glycosylation markers in HCC.


Assuntos
Carcinoma Hepatocelular/química , Glicômica/métodos , Neoplasias Hepáticas/química , Polissacarídeos/análise , Biomarcadores Tumorais/análise , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia de Fase Reversa , Glicosilação , Células Hep G2 , Humanos , Glicoproteínas de Membrana/análise , Polissacarídeos/química , Espectrometria de Massas em Tandem
15.
Rapid Commun Mass Spectrom ; 32(23): 2031-2039, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30152909

RESUMO

RATIONALE: The functional study and application of an intact glycoprotein require the structural characterization of both the protein backbone and the glycan moiety; the former has been successfully demonstrated with selective fragmentation of the protein backbone in CID and ExD; whether the latter can be achieved with selective fragmentation of the glycan moiety remains to be explored. METHODS: RNase B solution was electrosprayed and its intact glycoforms of GlcNAc2 Mann (n = 5-9) with the highest abundance (charge state z = 16) were isolated individually and fragmented using CID, ETD, HCD, ETciD, and EThcD on the Orbitrap Fusion Lumos Tribrid mass spectrometer; the dissociation parameters were optimized for selective fragmentation of the N-glycan moiety and protein backbone as well as high sequence coverage. The obtained spectra were interpreted using the protein and N-glycan database search engines ProteinGoggle and GlySeeker, respectively. RESULTS: With exploration of different dissociation parameters for all the five methods, selective fragmentation of the N-glycan moiety (the protein backbone staying intact) was observed in both HCD and EThcD at low collisional energies, but only a few matched product ions were observed; more comprehensive fragmentation was observed at high collisional energies (the protein backbone lost). Selective protein backbone fragmentation was observed in all the five dissociation methods. CONCLUSIONS: For comprehensive structural characterization of intact N-glycoproteins using tandem mass spectrometry, the composition and topology of the N-glycan moiety can be identified using HCD and EThcD complementarily at low and high energies; while the amino acid sequence and glycosite can be identified using CID, ETD, HCD, ETciD, and EThcD with their optimal dissociation parameters.


Assuntos
Ribonucleases/química , Motivos de Aminoácidos , Bases de Dados de Proteínas , Glicoproteínas/química , Glicosilação , Espectrometria de Massas em Tandem
16.
J Proteomics ; 184: 34-38, 2018 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-29935335

RESUMO

Core histones and their PTMs play important roles in regulating gene transcription and other DNA-related processes. The study of core histones PTMs, their cross-talk and functional roles is not only of broad biological significance but also of wide pathological and clinical relevance. Having the strength of comprehensive proteoform identification with 100% amino acid sequence coverage and combinatorial PTMs, top-down proteomics has become the state-of-the-art analytical tool for combinatorial PTM characterization of core histones. In this study, we report our top-down characterization of chicken (Gallus gallus domesticus) core histones, which have been widely used as models for chromosome re-construction among others because of easy availability and not-so-dense PTMs. With nanoRPLC-MS/MS analysis and ProteinGoggle database search, a total of 58 proteoforms were identified for the core histone families of H4, H2B, H2A, and H3.


Assuntos
Galinhas/metabolismo , Histonas/análise , Histonas/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Animais , Metilação , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem
17.
Anal Bioanal Chem ; 410(17): 4195-4202, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29725729

RESUMO

Aberrant glycosylation has been commonly observed in various physiological and pathological disorders (including cancers), and quite a few glycoproteins have been approved by the US Food and Drug Administration (FDA) as markers for early diagnosis. Each glycoprotein may have multiple glycoforms, and cancer-related ones can be only some specific glycoforms which have much higher sensitivity and specificity; for example, AFP glycoform AFP-L3 with N-glycan of 01Y(61F)41Y41M(31M41Y41L41S61M41Y41L41S is of bigger diagnostic value for hepatocellular carcinoma than total AFP (i.e., combination of all glycoforms). Mass spectrometry-based glycomics is currently the state-of-the-art instrumental analytical pipeline for high-throughput characterization of various glycoforms, where not only monosaccharide composition but also comprehensive structural information (sequence and linkage) of N-glycans are now reported thanks to our recently developed N-glycan database search engine GlySeeker. With this new capability, here, we report our large-scale characterization of human liver N-glycome with primary structures; 214 unique N-glycans with unique primary structures were identified and visualized with spectrum-level false discovery rate ≤ 1% and number of best hits of 1. The LO2 N-glycans reported here serve as a basic reference for future liver N-glycome study, and further quantitative analysis will enable characterization of differentially expressed N-glycans and discovery of more effective markers for liver and other diseases. Data are available via ProteomeXchange with identifier PXD008158.


Assuntos
Carcinoma Hepatocelular/patologia , Glicômica , Neoplasias Hepáticas , Fígado/patologia , Contagem de Células , Glicosilação , Humanos , Espectrometria de Massas
18.
Rapid Commun Mass Spectrom ; 32(2): 142-148, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29105226

RESUMO

RATIONALE: Most of the current popular tandem mass spectrometers have the capability of resolving the primary structures (monosaccharide composition, sequence and linkage) of N-glycans; however, compositions or putative structures have mostly been reported so far. Identification and visualization tools of N-glycans are needed. METHODS: The isotopic mass-to-charge ratio and envelope fingerprinting algorithm, which has been successfully used for intact protein database search and identification, was adapted for N-glycan database search, and a stand-alone N-glycan database search engine, GlySeeker, for automated N-glycan identification and visualization was developed and successfully benchmarked. Both pseudo 2D graph and one-line text formats with one-letter symbols for monosaccharides were proposed for representing N-glycans. N-glycans were identified with comprehensive interpretation of product ions and false discovery rate (FDR) control. RESULTS: In a database search of reversed-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) datasets of the N-glycome enriched from OVCAR-3 ovarian cancer cells, with FDR ≤1% and number of best hits (NoBHs) = 1-30, 1525 N-glycans with comprehensive primary structural information (composition, sequence and linkage) were identified and visualized; among these 1525 N-glycans, 559 had NoBHs = 1, i.e. their structures were uniquely identified. This represents a large-scale identification and visualization of N-glycans with primary structures from tandem mass spectra. CONCLUSIONS: A stand-alone N-glycan database search engine called GlySeeker has been developed for large-scale identification and visualization of N-glycans with comprehensive interpretation of tandem mass spectra and FDR control.

19.
Anal Chim Acta ; 996: 38-47, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29137706

RESUMO

Phosphorylation is one of the most important and widely studied protein post-translational modifications. Tandem mass spectrometry using higher-energy collisional dissociation has evolved into a state-of-the-art analytical platform for both phosphorylation identification and site localization. Tens of thousands of phosphopeptides can now be routinely identified from a single shotgun proteomics study; site localization, however, is much more complicated and many challenges still exist. Here, we report our development of P-bracket using direct experimental evidence of phospho-containing site-determining product ions for accurate site localization without the need for additional FLR control. A P-bracket is defined as a complementary product ion pair that forms a bracket to confine a phosphorylation event to a unique site. P-bracket has been successfully benchmarked with a set of six synthetic phosphopeptides with a single phosphorylation event, a set of 96 synthetic peptides and phosphopeptide reference libraries, and two HeLa phosphopeptide LC-MS/MS (HCD) datasets; Accurate phosphosite localization by P-bracket will greatly enhance identification confidence of phosphopeptides and contribute to structural and functional studies of phosphoproteins.


Assuntos
Fosfopeptídeos/química , Fosforilação , Proteômica , Técnicas de Química Sintética , Cromatografia Líquida , Células HeLa , Humanos , Biblioteca de Peptídeos , Espectrometria de Massas em Tandem
20.
J Proteomics ; 160: 21-27, 2017 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-28336331

RESUMO

Neutral loss and internal product ions have been found to be significant in both peptide and protein tandem mass spectra and they have been proposed to be included in database search and for protein identification. In addition to common canonical b/y ions in collision-based dissociation or c/z ions in electron-based dissociation, inclusion of neutral loss and internal product ions would certainly make better use of tandem mass spectra data; however, their ultimate utility for protein identification with false discovery rate control remains unclear. Here we report our proteome-level utility benchmarking of neutral loss and internal product ions with tandem mass spectra of intact E. coli proteome. Utility of internal product ions was further evaluated at the protein level using selected tandem mass spectra of individual E. coli proteins. We found that both neutral loss and internal products ions do not have direct utility for protein identification when they were used for scoring of P Score; but they do have indirect utility for provision of more canonical b/y ions when they are included in the database search and overlapping ions between different ion types are resolved. BIOLOGICAL SIGNIFICANCE: Tandem mass spectrometry has evolved to be a state-of-the-art method for characterization of protein primary structures (including amino acid sequence, post-translational modifications (PTMs) as well as their site location), where full study and utilization tandem mass spectra and product ions are indispensable. This primary structure information is essential for higher order structure and eventual function study of proteins.


Assuntos
Escherichia coli/química , Proteoma/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Bases de Dados de Proteínas/normas , Proteínas de Escherichia coli/análise , Íons , Peptídeos/análise , Processamento de Proteína Pós-Traducional , Proteínas/análise
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