Specific isolation of N-terminal fragments from proteins and their high-fidelity de novo sequencing.
A new method to determine N-terminal amino acid sequences of multiple proteins at low pmol level by a parallel processing has been developed. The method contains the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling with sulfosucccimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate(sulfo-NHS-SS-biotin) to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease; (4) specific isolation of N-terminal fragments of proteins by affinity capture using the biotin-avidin system; (5) de novo sequence analysis of peptides by MALDI-TOF-/MALDI-TOF-PSD mass spectrometry with effective utilization of the CAF (chemically assisted fragmentation) method.1 This method is also effective for N-terminal sequencing of each protein in a mixture of several proteins, and for sequencing components of a multiprotein complex. It is expected to become an essential proteomics tool for identifying proteins, especially when used in combination with a C-terminal sequencing method.
Yamaguchi
Minoru
M
Life Science Laboratory, Shimadzu Corporation, Kyoto 604-8511, Japan. yamagu@shimadzu.co.jp
Obama
Takashi
T
Kuyama
Hiroki
H
Nakayama
Daisuke
D
Ando
Eiji
E
Okamura
Taka-Aki
TA
Ueyama
Norikazu
N
Nakazawa
Takashi
T
Norioka
Shigemi
S
Nishimura
Osamu
O
Tsunasawa
Susumu
S
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001426:Bacterial Proteins; D010446:Peptide Fragments