A method for rapidly confirming protein N-terminal sequences by matrix-assisted laser desorption/ionization mass spectrometry.
The N-terminal sequence is important for the identification of a protein and the confirmation of its N-terminal processing. Although mass spectrometry (MS) is a sensitive and high-throughput method to sequence and identify peptides and proteins, N-terminal peptides, diluted among most of the peptides that do not originate at the N-termini, are not easy to identify directly with MS. To develop a simple and rapid method to identify and sequence the N-terminal peptide of a protein, a new strategy based on specific sulfonation of terminal amino groups and selective monitoring of the sulfonated peptide was introduced. After a protein had been guanidinated, 2-sulfobenzoylated, and reduced, it was digested with trypsin and analyzed by MS. Because of the strong acidity of sulfonic groups and the specific sulfonation of alpha-amino groups, the sulfonated N-terminal peptide dominated as base peak in the negative mode peptide mass fingerprint (PMF) and was easy to identify. The N-terminal peptide was then selected as precursor ion for tandem mass spectrometric (MS/MS) analysis. Four proteins were tested with this method and their N-terminal peptides were successfully recognized and sequenced. The results suggest that the addition of a sulfonic acid group facilitates the identification and de novo sequencing of N-terminal peptides.
Zhou
Chunxi
C
Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, 27 Tai-Ping Road, Beijing 100850, China.
Zhang
Yangjun
Y
Qin
Peibin
P
Liu
Xin
X
Zhao
Liyan
L
Yang
Shuhua
S
Cai
Yun
Y
Qian
Xiaohong
X
D000818:Animals; D002645:Chickens; D006736:Horses; D019382:Human Growth Hormone / Q000737:chemistry; D006801:Humans; D009113:Muramidase / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D009211:Myoglobin; D011506:Proteins; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D009113:Muramidase