193 nm Ultraviolet Photodissociation of Imidazolinylated Lys-N Peptides for De Novo Sequencing

• Published in: Analytical chemistry (Washington) Vol. 84; no. 5; pp. 2433 - 2439
• Main Authors: Robinson, Michelle R, Madsen, James A, Brodbelt, Jennifer S
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 06-03-2012
• Subjects: Amino Acid Sequence; Analytical chemistry; Chemistry; Exact sciences and technology; Imidazoles - chemistry; Ions; Lysine - chemistry; Peptides; Peptides - chemistry; Photolysis; Sequence Analysis, Protein; Spectrometric and optical methods; Tandem Mass Spectrometry; Ultraviolet radiation; Ultraviolet Rays; Peptides; Enzyme; Use; Product; Identification; Method; Photodissociation; Precursor; De novo; Peptidases; Lysine; Residue; Mass spectrometry MS/MS; Hydrolases; Strategy; Diagnosis; Sequencing
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac203227y

The goal of many MS/MS de novo sequencing strategies is to generate a single product ion series that can be used to determine the precursor ion sequence. Most methods fall short of achieving such simplified spectra, and the presence of additional ion series impede peptide identification. The present study aims to solve the problem of confounding ion series by enhancing the formation of “golden” sets of a, b, and c ions for sequencing. Taking advantage of the characteristic mass differences between the golden ions allows N-terminal fragments to be readily identified while other ion series are excluded. By combining the use of Lys-N, an alternate protease, to produce peptides with lysine residues at each N-terminus with subsequent imidazolinylation of the ε-amino group of each lysine, peptides with highly basic sites localized at each N-terminus are generated. Subsequent MS/MS analysis by using 193 nm ultraviolet photodissociation (UVPD) results in enhanced formation of the diagnostic golden pairs and golden triplets that are ideal for de novo sequencing.