Screening for disulfide-rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57 u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116 u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate.
Neitz
S
S
BioVisioN GmbH & Co. KG, Feodor-Lynen-Str. 5, 30625 Hannover, Germany.
Jürgens
M
M
Kellmann
M
M
Schulz-Knappe
P
P
Schrader
M
M
D000588:Amines / Q000737:chemistry*; D002264:Carboxylic Acids / Q000737:chemistry*; D003545:Cysteine / Q000032:analysis; D004220:Disulfides / Q000032:analysis* / Q000097:blood; D006801:Humans; D008745:Methylation; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000097:blood; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000588:Amines; D002264:Carboxylic Acids; D004220:Disulfides; D010455:Peptides; D003545:Cysteine