LC-mass spectrometry analysis of N- and C-terminal boundary sequences of polypeptide fragments by limited proteolysis

• Published in: Journal of the American Society for Mass Spectrometry Vol. 16; no. 1; pp. 38 - 45
• Main Authors: Stroh, Justin G., Loulakis, Pat, Lanzetti, Anthony J., Xie, Julie
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 2005; Elsevier Science; Springer Nature B.V
• Subjects: Activin Receptors - chemistry; Amino Acid Sequence; Aminoacids, peptides. Hormones. Neuropeptides; Analytical chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, High Pressure Liquid; Enzymes; Exact sciences and technology; Fragments; Fundamental and applied biological sciences. Psychology; Mass spectrometry; Molecular Sequence Data; Molecular Weight; Other chromatographic methods; Peptide Fragments - chemistry; Peptide Mapping; Protein Structure, Tertiary; Proteins; Recognition; Scientific imaging; Sensitivity analysis; Spectrometry, Mass, Electrospray Ionization - methods; Spectroscopy; Thermolysin - chemistry; Chemical analysis; Serine endopeptidases; Peptides; Molecular structure; Enzyme; Coupled method; HPLC chromatography; Metalloendopeptidases; Thermolysin; Electrospray; C terminal-Sequence; Peptidases; Peptide map; Endopeptidase K; Peptide fragment; Polypeptide; Proteolysis; N terminal-Sequence; Hydrolases; Degradation product; Mass spectrometry; Aminoacid sequence; Structural analysis
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/j.jasms.2004.08.018

Limited proteolysis is an important and widely used method for analyzing the tertiary structure and determining the domain boundaries of proteins. Here we describe a novel method for determining the N- and C-terminal boundary amino acid sequences of products derived from limited proteolysis using semi-specific and/or non-specific enzymes, with mass spectrometry as the only analytical tool. The core of this method is founded on the recognition that cleavage of proteins with non-specific proteases is not random, but patterned. Based on this recognition, we have the ability to determine the sequence of each proteolytic fragment by extracting a common association between data sets containing multiple potential sequences derived from two or more different mass spectral molecular weight measurements. Proteolytic product sequences derived from specific and non-specific enzymes can be accurately determined without resorting to the conventional time-consuming and laborious methods of SDS-PAGE and N-terminal sequencing analysis. Because of the sensitivity of mass spectrometry, multiple transient proteolysis intermediates can also be identified and analyzed by this method, which allows the ability to monitor the progression of proteolysis and thereby gain insight into protein structures.