De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature.
De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.
Department of Chemistry, Seoul National University, Seoul 151-742, Republic of Korea. jongseo.kim1@gmail.com
Jong-Seo
JS
Kim
Jin-Su
JS
Song
Yongju
Y
Kim
Seung Bum
SB
Park
Hie-Joon
HJ
Kim
D000595:Amino Acid Sequence; D001966:Bromine / Q000737:chemistry*; D002918:Chymotrypsin / Q000737:chemistry; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry; D007096:Imidoesters / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D006454:Hemoglobins; D007096:Imidoesters; D009211:Myoglobin; D010748:Phosphopeptides; D011506:Proteins; D002918:Chymotrypsin; D008239:Lysine; D001966:Bromine