Automated online sequential isotope labeling for protein quantitation applied to proteasome tissue-specific diversity.
Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic labeling. To analyze tissue-derived samples, the isotopic labeling can be performed using chemical labeling of the peptides post-digestion. Standard chemical labeling procedures often require many manual sample handling steps, reducing the accuracy of measurements. Here, we describe a fully automated, online (in nanoLC columns), labeling procedure, which allows protein quantitation using differential isotopic dimethyl labeling of peptide N termini and lysine residues. We show that the method allows reliable quantitation over a wide dynamic range and can be used to quantify differential protein abundances in lysates and, more targeted, differences in composition between purified protein complexes. We apply the method to determine the differences in composition between bovine liver and spleen 20 S core proteasome complexes. We find that although all catalytically active immunoproteasome subunits were up-regulated in spleen (compared with liver), only one of the normal catalytic subunits was down-regulated, suggesting that the tissue-specific immunoproteasome assembly is more diverse than previously assumed.
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Reinout
R
Raijmakers
Celia R
CR
Berkers
Annemieke
A
de Jong
Huib
H
Ovaa
Albert J R
AJ
Heck
Shabaz
S
Mohammed
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D007553:Isotope Labeling / Q000379:methods*; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D009862:Online Systems; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000032:analysis; D013154:Spleen / Q000201:enzymology
D011506:Proteins; D012710:Serum Albumin, Bovine; D046988:Proteasome Endopeptidase Complex; D008239:Lysine