Probing adenosine nucleotide-binding proteins with an affinity-labeled nucleotide probe and mass spectrometry.
Mass spectrometry combined with chemical labeling strategies has become very important in biological analysis. Herein, we described the application of a biotin-conjugated acyl nucleotide for probing adenosine nucleotide-binding proteins. We demonstrated that the probe reacted specifically with the lysine residue at the nucleotide-binding site of two purified adenosine nucleotide-binding proteins, Escherichia coli recombinase A (RecA) and Saccharomyces cerevisiae alcohol dehydrogenase-I (YADH-I). A single conjugate peptide with a specifically labeled lysine residue was identified, by using LC-MS/MS, from the tryptic digestion mixture of the reaction products of the nucleotide analogue with RecA or YADH-I. The strategy, which involved labeling reaction, enzymatic digestion, affinity purification, and LC-MS/MS analysis, was relatively simple, fast, and straightforward. The method should be generally applicable for the identification of lysine residues at the nucleotide-binding site of other proteins. The biotin-conjugated acyl nucleotide probe also allowed for the enrichment and identification of nucleotide-binding proteins from complex protein mixtures; we showed that more than 50 adenosine nucleotide-binding proteins could be identified from the whole-cell lysates of HeLa-S3 and WM-266-4 cells.
Qiu
Haibo
H
Department of Chemistry-027, University of California, Riverside, California 92521-0403, USA.
Wang
Yinsheng
Y
D000241:Adenosine / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000345:Affinity Labels; D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001710:Biotin / Q000032:analysis / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007553:Isotope Labeling; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000032:analysis*; D011938:Rec A Recombinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D015335:Molecular Probes; D009711:Nucleotides; D001710:Biotin; D000255:Adenosine Triphosphate; D000426:Alcohol Dehydrogenase; D011938:Rec A Recombinases; D014357:Trypsin; D008239:Lysine; D000241:Adenosine