Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.
Article Details
- CitationCopy to clipboard
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
Name UniProt ID Tubulin beta-1 chain Q9H4B7 Details Phenylalanine--tRNA ligase alpha subunit Q9Y285 Details Transketolase P29401 Details Delta-aminolevulinic acid dehydratase P13716 Details Pyruvate kinase PKM P14618 Details Adenine phosphoribosyltransferase P07741 Details Hemoglobin subunit alpha P69905 Details NAD-dependent malic enzyme, mitochondrial P23368 Details Tyrosine--tRNA ligase, cytoplasmic P54577 Details Sorbitol dehydrogenase Q00796 Details Transthyretin P02766 Details Isovaleryl-CoA dehydrogenase, mitochondrial P26440 Details Serum albumin P02768 Details NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 Q86Y39 Details Glyceraldehyde-3-phosphate dehydrogenase P04406 Details C-1-tetrahydrofolate synthase, cytoplasmic P11586 Details Peptidyl-prolyl cis-trans isomerase FKBP1A P62942 Details Glutathione S-transferase P P09211 Details Platelet glycoprotein IX P14770 Details L-xylulose reductase Q7Z4W1 Details Protein kinase C beta type P05771 Details Mitogen-activated protein kinase 1 P28482 Details Platelet basic protein P02775 Details Spermine synthase P52788 Details Peptidyl-prolyl cis-trans isomerase A P62937 Details Mitogen-activated protein kinase 14 Q16539 Details Platelet glycoprotein Ib alpha chain P07359 Details von Willebrand factor P04275 Details Glucose-6-phosphate 1-dehydrogenase P11413 Details Protein kinase C alpha type P17252 Details Integrin beta-3 P05106 Details Endoplasmin P14625 Details 14-3-3 protein zeta/delta P63104 Details Catalase P04040 Details Histidine triad nucleotide-binding protein 1 P49773 Details Fructose-bisphosphate aldolase A P04075 Details Macrophage migration inhibitory factor P14174 Details Fatty acid-binding protein, epidermal Q01469 Details Fibrinogen beta chain P02675 Details Protein ERGIC-53 P49257 Details Glycogen phosphorylase, brain form P11216 Details Fatty-acid amide hydrolase 1 O00519 Details Glutamyl-tRNA(Gln) amidotransferase subunit B, mitochondrial O75879 Details N-acetyl-D-glucosamine kinase Q9UJ70 Details Protein disulfide-isomerase P07237 Details Wiskott-Aldrich syndrome protein P42768 Details Annexin A3 P12429 Details Amyloid beta A4 protein P05067 Details cAMP-dependent protein kinase type I-alpha regulatory subunit P10644 Details Apolipoprotein A-I P02647 Details Gelsolin P06396 Details Destrin P60981 Details Cofilin-1 P23528 Details Actin-related protein 2/3 complex subunit 1B O15143 Details Actin-related protein 2/3 complex subunit 2 O15144 Details Actin-related protein 2/3 complex subunit 3 O15145 Details Actin-related protein 2/3 complex subunit 4 P59998 Details Actin-related protein 2/3 complex subunit 5 O15511 Details Thiosulfate sulfurtransferase Q16762 Details Acyl-CoA-binding protein P07108 Details Galectin-1 P09382 Details Elongation factor Tu, mitochondrial P49411 Details 14-3-3 protein epsilon P62258 Details Transitional endoplasmic reticulum ATPase P55072 Details Sorting nexin-3 O60493 Details Eukaryotic translation initiation factor 2 subunit 3 P41091 Details Peroxiredoxin-6 P30041 Details Keratin, type I cytoskeletal 12 Q99456 Details Actin, cytoplasmic 1 P60709 Details Actin, cytoplasmic 2 P63261 Details Lysosomal protective protein P10619 Details 60 kDa heat shock protein, mitochondrial P10809 Details Alpha-actinin-1 P12814 Details 14-3-3 protein beta/alpha P31946 Details Protein disulfide-isomerase A3 P30101 Details RuvB-like 2 Q9Y230 Details Tropomyosin alpha-4 chain P67936 Details 14-3-3 protein eta Q04917 Details 14-3-3 protein theta P27348 Details Alpha-2-HS-glycoprotein P02765 Details Keratin, type II cytoskeletal 1 P04264 Details ATP synthase subunit O, mitochondrial P48047 Details Transgelin-2 P37802 Details Calcium/calmodulin-dependent protein kinase type 1 Q14012 Details Tyrosine-protein phosphatase non-receptor type 12 Q05209 Details Eukaryotic translation initiation factor 4E-binding protein 1 Q13541 Details