title: Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis.
abstract: The characterization of protein modifications is essential for the study of protein function using functional genomic and proteomic approaches. However, current techniques are not efficient in determining protein modifications. We report an approach for sequencing proteins and determining modifications with high speed, sensitivity and specificity. We discovered that a protein could be readily acid-hydrolyzed within 1 min by exposure to microwave irradiation to form, predominantly, two series of polypeptide ladders containing either the N- or C-terminal amino acid of the protein, respectively. Mass spectrometric analysis of the hydrolysate produced a simple mass spectrum consisting of peaks exclusively from these polypeptide ladders, allowing direct reading of amino acid sequence and modifications of the protein. As examples, we applied this technique to determine protein phosphorylation sites as well as the sequences and several previously unknown modifications of 28 small proteins isolated from Escherichia coli K12 cells. This technique can potentially be automated for large-scale protein annotation.
authors.forename: Hongying
authors.initials: H
authors.lastname: Zhong
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Wen
authors.forename: Liang
authors.initials: L
authors.lastname: Li
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence / Q000528:radiation effects; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods*; D008872:Microwaves; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D020539:Sequence Analysis, Protein / Q000379:methods*
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins