STRUCTURAL-ANALYSIS OF PROTEINS BY CAPILLARY HPLC ELECTROSPRAY TANDEM MASS-SPECTROMETRY

Capillary high performance liquid chromatography (HPLC) with on-line ultraviolet detection was coupled with electrospray tandem mass spectrometry. Protein tryptic digests were analyzed at sample levels as low as 800 fmol. Collision-activated dissociation was used to obtain amino acid sequence information on peptides at the 5 pmol level. Protein spotted on to nitrocellulose membrane was digested with trypsin and the resultant peptide mixture was analyzed by capillary HPLC-MS. A deoxynucleotide binding protein isolated by affinity chromatography and sodium dodecylsulfate-polyacrylamide gel electrophoresis was digested in situ and analyzed using this system. The methodology presented in this paper offers a high sensitivity, high throughput strategy for analyzing the primary structure of proteins.