Profiling constitutive proteolytic events in vivo.
Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.
Timmer
John C
JC
Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Enoksson
Mari
M
Wildfang
Eric
E
Zhu
Wenhong
W
Igarashi
Yoshinobu
Y
Denault
Jean-Benard
JB
Ma
Yuliang
Y
Dummitt
Benjamin
B
Chang
Yie-Hwa
YH
Mast
Alan E
AE
Eroshkin
Alexey
A
Smith
Jeffrey W
JW
Tao
W Andy
WA
Salvesen
Guy S
GS
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D007611:Aprotinin / Q000737:chemistry / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D020169:Caspases / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D016681:Genome, Fungal; D006801:Humans; D063208:Methionyl Aminopeptidases; D008782:Methylurea Compounds / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001798:Blood Proteins; D008782:Methylurea Compounds; D010455:Peptides; D020543:Proteome; C002249:O-methylisourea; D007611:Aprotinin; D010447:Peptide Hydrolases; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; D020169:Caspases