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...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 1995-11, Vol.67 (21), p.3971-3978
Main Authors: Patterson, Dale H, Tarr, George E, Regnier, Fred E, Martin, Stephen A
Format: Article
Language:English
Subjects:
Adrenocorticotropic Hormone - chemistry
Adrenocorticotropic Hormone - metabolism
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Carboxypeptidases - chemistry
Carboxypeptidases - metabolism
Cathepsin A
Chemistry
Dose-Response Relationship, Drug
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods
Hormone Antagonists - chemistry
Hormone Antagonists - metabolism
Humans
Molecular Sequence Data
Peptide Fragments - chemistry
Peptide Fragments - metabolism
Proteins
Scientific imaging
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac00117a024