Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

Article Details

Citation

Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR, Vandekerckhove J

Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

Nat Biotechnol. 2003 May;21(5):566-9. Epub 2003 Mar 31.

PubMed ID
12665801 [ View in PubMed
]
Abstract

Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
Tubulin beta-1 chainQ9H4B7Details
Phenylalanine--tRNA ligase alpha subunitQ9Y285Details
TransketolaseP29401Details
Delta-aminolevulinic acid dehydrataseP13716Details
Pyruvate kinase PKMP14618Details
Adenine phosphoribosyltransferaseP07741Details
Hemoglobin subunit alphaP69905Details
NAD-dependent malic enzyme, mitochondrialP23368Details
Tyrosine--tRNA ligase, cytoplasmicP54577Details
Sorbitol dehydrogenaseQ00796Details
TransthyretinP02766Details
Isovaleryl-CoA dehydrogenase, mitochondrialP26440Details
Serum albuminP02768Details
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11Q86Y39Details
Glyceraldehyde-3-phosphate dehydrogenaseP04406Details
C-1-tetrahydrofolate synthase, cytoplasmicP11586Details
Peptidyl-prolyl cis-trans isomerase FKBP1AP62942Details
Glutathione S-transferase PP09211Details
Platelet glycoprotein IXP14770Details
L-xylulose reductaseQ7Z4W1Details
Protein kinase C beta typeP05771Details
Mitogen-activated protein kinase 1P28482Details
Platelet basic proteinP02775Details
Spermine synthaseP52788Details
Peptidyl-prolyl cis-trans isomerase AP62937Details
Mitogen-activated protein kinase 14Q16539Details
Platelet glycoprotein Ib alpha chainP07359Details
von Willebrand factorP04275Details
Glucose-6-phosphate 1-dehydrogenaseP11413Details
Protein kinase C alpha typeP17252Details
Integrin beta-3P05106Details
EndoplasminP14625Details
14-3-3 protein zeta/deltaP63104Details
CatalaseP04040Details
Histidine triad nucleotide-binding protein 1P49773Details
Fructose-bisphosphate aldolase AP04075Details
Macrophage migration inhibitory factorP14174Details
Fatty acid-binding protein, epidermalQ01469Details
Fibrinogen beta chainP02675Details
Protein ERGIC-53P49257Details
Glycogen phosphorylase, brain formP11216Details
Fatty-acid amide hydrolase 1O00519Details
Glutamyl-tRNA(Gln) amidotransferase subunit B, mitochondrialO75879Details
N-acetyl-D-glucosamine kinaseQ9UJ70Details
Protein disulfide-isomeraseP07237Details
Wiskott-Aldrich syndrome proteinP42768Details
Annexin A3P12429Details
Amyloid beta A4 proteinP05067Details
cAMP-dependent protein kinase type I-alpha regulatory subunitP10644Details
Apolipoprotein A-IP02647Details
GelsolinP06396Details
DestrinP60981Details
Cofilin-1P23528Details
Actin-related protein 2/3 complex subunit 1BO15143Details
Actin-related protein 2/3 complex subunit 2O15144Details
Actin-related protein 2/3 complex subunit 3O15145Details
Actin-related protein 2/3 complex subunit 4P59998Details
Actin-related protein 2/3 complex subunit 5O15511Details
Thiosulfate sulfurtransferaseQ16762Details
Acyl-CoA-binding proteinP07108Details
Galectin-1P09382Details
Elongation factor Tu, mitochondrialP49411Details
14-3-3 protein epsilonP62258Details
Transitional endoplasmic reticulum ATPaseP55072Details
Sorting nexin-3O60493Details
Eukaryotic translation initiation factor 2 subunit 3P41091Details
Peroxiredoxin-6P30041Details
Keratin, type I cytoskeletal 12Q99456Details
Actin, cytoplasmic 1P60709Details
Actin, cytoplasmic 2P63261Details
Lysosomal protective proteinP10619Details
60 kDa heat shock protein, mitochondrialP10809Details
Alpha-actinin-1P12814Details
14-3-3 protein beta/alphaP31946Details
Protein disulfide-isomerase A3P30101Details
RuvB-like 2Q9Y230Details
Tropomyosin alpha-4 chainP67936Details
14-3-3 protein etaQ04917Details
14-3-3 protein thetaP27348Details
Alpha-2-HS-glycoproteinP02765Details
Keratin, type II cytoskeletal 1P04264Details
ATP synthase subunit O, mitochondrialP48047Details
Transgelin-2P37802Details
Calcium/calmodulin-dependent protein kinase type 1Q14012Details
Tyrosine-protein phosphatase non-receptor type 12Q05209Details