C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions.
The utility of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the analysis of C-terminal peptide ladders from carboxypeptidase Y (CPY) digestions is discussed. MALDI analysis of aliquots of an optimized time-dependent CPY digestion of ACTH 7-38 fragment allowed for the sequence of the first 19 amino acids from the C-terminus to be determined in 25 min of digestion time. A strategy for performing parallel concentration-dependent digestions on the MAL-DI plate is proven to be superior to the time-dependent approach as the method development time and practical amounts of both peptide and enzyme consumed are reduced significantly. The on-plate approach offered the same sequence information from the ACTH 7-38 fragment and was used to digest 22 peptides of various amino acid composition, size, charge, and polarity. Of the 22 peptides digested on-plate, sequence information was derived from 19 of them. A statistical analysis strategy for ladder sequencing utilizing t-statistics is offered as a method for placing confidence intervals on residue assignments.
Patterson
D H
DH
PerSeptive Biosystems, Framingham, Massachusetts 01701, USA.
Tarr
G E
GE
Regnier
F E
FE
Martin
S A
SA
D000324:Adrenocorticotropic Hormone / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism*; D043402:Cathepsin A; D004305:Dose-Response Relationship, Drug; D006727:Hormone Antagonists / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006727:Hormone Antagonists; D010446:Peptide Fragments; C026034:ACTH (7-38); D000324:Adrenocorticotropic Hormone; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A