Electrophoresis combined with novel mass spectrometry techniques: Powerful tools for the analysis of proteins and proteomes

Figeys, Daniel; Gygi, Steven P.; Zhang, Yanni; Watts, Julian D.; Gu, Min; Aebersold, Ruedi

doi:10.1002/elps.1150191045

Cited by 88 publications

(55 citation statements)

References 44 publications

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“…Magnetic sector Sheathless [164] Sequence analysis of peptides MPC-CZE Magnetic sector Sheath liquid [165] Peptide standards SPE-CZE Ion trap Sheathless [166] Proteins and proteomes SPE-CZE Sheathless [167] Proteolytic digests CZE Triple quadrupole, QqTOF Sheath liquid [168] Neurotransmitters and neuropeptides CZE Triple quadrupole Sheath liquid [169] Metallothioneins CZE Triple quadrupole Sheathless [170] Protein standards CZE Single quadrupole Sheath liquid [171] Analysis of proteins from SDS-PAGE gels CZE IT-reTOF-MS Sheathless [172] Neuropeptide Y, its fragments and two of their degradation products CZE Sheath liquid [173] Proteins in complexes, proteolytic digests CZE/micro-SPE Ion trap Liquid junction [174] Phosphopeptides IMAC-CZE c) Ion trap In-column electrode [175] Reduced and oxidized forms of cytochrome c [176] Phospholipase glycosylation patterns of bee venoms CD-CZE Ion trap Sheath liquid, sheathless [177] Gel-isolated membrane proteins CZE Triple quadrupole, QqTOF Sheathless [178] Peptide standards CZE TOF Sheathless [179] 20 natural amino acids in children's blood CZE Tandem quadrupole Sheath liquid [180] Evaluation of immobilized trypsin beds for on-line digestion of proteins CZE Single quadrupole Sheathless [181] Protein profiles of Siberian permafrost bacteria CZE IT-reTOF [182] Phosphorylation sites of proteins IMAC-CZE Ion trap [183] Protein standards TITP Single quadrupole Sheath liquid [183] Cytochrome c digests and a peptidomimetic direct thrombin inhibitor TITP Single quadrupole Sheath liquid [184] Bacterial proteomes CIEF FTICR Sheath liquid [185] Histones CZE Sheath liquid [186] Metal displacement of a mouse liver metallothionein CZE Triple quadrupole Sheath liquid [187] Protein standards in PEG-containing samples CZE Triple quadrupole Sheath liquid [188] Amyloid-b peptide CZE Single quadrupole Sheath liquid [189] Protein standards CZE Ion trap Sheath liquid [190] Peptide standards CZE Ion trap Sheath liquid …”

Section: ] Capillary Isotachophoresis Esi-ms (Citp-esi-ms) A)mentioning

confidence: 99%

Capillary electrophoresis – mass spectrometry: 15 years of developments and applications

Schmitt‐Kopplin¹,

Frommberger²

2003

Electrophoresis

282

232

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Capillary electrophoresis -mass spectrometry: 15 years of developments and applicationsSince its introduction in 1987, capillary electrophoresis-mass spectrometry (CE-MS) has developed to a well accepted multidimensional analytical approach complementary and/or competitive to classical MS-hyphenated separation techniques. The threefold combination of rapid developments of an exceptional separation technique, of selective mass detection possibilities, and of very mild ionization modes first allowed these progresses. This article shows the CE specificities that need to be well controlled/known, compared to classical and more routinely used liquid chromatography in the light of its coupling to MS. The major trends and developments over the last 15 years and most of the reviews and applications found in ISI Web of science and publisher databases are presented in a tabulated way. The reader can thus rapidly find existing CE-MS analysis techniques in his field of research and application (forensics, environment, bioanalytics, pharmaceutics, and metabolites).

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Section: ] Capillary Isotachophoresis Esi-ms (Citp-esi-ms) A)mentioning

confidence: 99%

Capillary electrophoresis – mass spectrometry: 15 years of developments and applications

Schmitt‐Kopplin¹,

Frommberger²

2003

Electrophoresis

282

232

View full text Add to dashboard Cite

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“…Moreover, there have been no other reports elucidating the antigens against AECA in BD. The human genome was completely sequenced recently, and as a result, many studies analyzing the function of various human genes are now (24)(25)(26).…”

Section: Aeca Against ␣-Enolase In Behç Et's Disease 2031mentioning

confidence: 99%

Human α‐enolase from endothelial cells as a target antigen of anti–endothelial cell antibody in Behçet's disease

Lee¹,

Chung²,

Kim³

et al. 2003

Arthritis & Rheumatism

175

108

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Objective. To identify and recombine a protein of the human dermal microvascular endothelial cell (HD-MEC) that specifically reacts with anti-endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD.Methods. The proteomics technique, with 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization؊time-of-flight (MALDI-TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme-linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis).Results. Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be ␣-enolase by 2-dimensional gel electrophoresis followed by immunoblotting and MALDI-TOF mass spectrometry. Recombinant ␣-enolase protein was isolated and refined by gene cloning. On Western blots, AECApositive IgM from the sera of patients with active BD reacted strongly with recombinant human ␣-enolase. BD patient sera positive for anti-␣-enolase did not react with human ␥-enolase. On dot-blotting, reactivity to human ␣-enolase was detected only in the IgM-positive group. Fifteen of the 18 AECA-positive sera that were positive for the HDMEC antigen showed reactivity to recombinant ␣-enolase IgM antibody by ELISA.Conclusion. The ␣-enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to ␣-enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, ␣-enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.

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“…Furthermore, because we sought to carry out relative quantitative measurements it was essential to preserve the ratios of modified to unmodified peptides that would be representative of any phosphorylation within the original protein. A phospho-enrichment procedure would negate this and would likely result in differential enrichment of the various phosphorylated species; therefore we avoided using enrichment procedures such as IMAC (immobilized metal-ion affinity chromatography) [32][33][34][35], strong cation exchange [36,37], or phospho-specific immunoprecipitation [38]. Using an optimized MS approach developed by monitoring known phosphorylation sites, Ser-118 and Ser-167, we demonstrated that Ser-154 in ER␣-NTD, long suspected but unconfirmed as being phosphorylated under endogenous intracellular conditions [27], is indeed partially phosphorylated under both ligand-dependent and ligand-independent human breast cancer growth conditions.…”

mentioning

confidence: 99%

A novel serine phosphorylation site detected in the n-terminal domain of estrogen receptor isolated from human breast cancer cells

Britton

Scott

Schilling

et al. 2008

J. Am. Soc. Mass Spectrom.

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Activated estrogen receptor (ER␣) plays a critical role in breast cancer development and is a major target for drug treatment. Serine phosphorylation within the N-terminal domain (NTD) contributes to ER␣ activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ER␣ residues was limited to protein artificially overexpressed in transfected cell lines. We report mass spectrometric methods that have allowed the identification of a new site within the NTD of ER␣ isolated from cultured human breast cancer cells. Immunoprecipitation, trypsin digestion, and analysis by nano-LC-ESI-MS/MS (Q-STAR, MDS Sciex) and vMALDI-MS n (Finnigan™ LTQ™, Thermo-Electron) identified peptides containing 8 of 14 serine residues within the NTD, one being partially phosphorylated Ser-167, known but not previously reported by MS. Chymotrypsin digestion revealed other known sites at Ser-102/104/106 and 118. Tandem methods developed for the peptide containing Ser-118 and the use of hypothesis-driven experiments-i.e., the assumption that an intact phosphopeptide showing no molecular ion might yield fragment ions including loss of phosphoric acid in vMALDI-MS/MS-allowed the identification of a novel site at Ser-154. Quantitation by selected reaction monitoring demonstrated 6-fold and 2.5-fold increases in Ser-154 phosphorylation in estradiol-and EGF-treated cells, respectively, compared to controls, confirmed by immunoblotting with a novel rabbit polyclonal antibody. Thus, the protein isolation and MS strategies described here can facilitate discovery of novel phosphorylation sites within low abundance, clinically important cancer targets like ER␣, and may thereby contribute to our understanding of the role of phosphorylation in the development of breast cancer. (J Am

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Electrophoresis combined with novel mass spectrometry techniques: Powerful tools for the analysis of proteins and proteomes

Cited by 88 publications

References 44 publications

Capillary electrophoresis – mass spectrometry: 15 years of developments and applications

Capillary electrophoresis – mass spectrometry: 15 years of developments and applications

Human α‐enolase from endothelial cells as a target antigen of anti–endothelial cell antibody in Behçet's disease

A novel serine phosphorylation site detected in the n-terminal domain of estrogen receptor isolated from human breast cancer cells

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