Simplification of high-energy collision spectra of peptides by amino-terminal derivatization.
Four-sector tandem mass spectrometry proves extremely useful for providing sequence information for peptides. The complexity of ion fragmentations, however, makes data interpretation difficult and time consuming. Attachment of a fixed positive charge to the peptide amino terminus forces production of only N-terminal fragment ions to yield simplified, predictable fragmentation. Reaction of a peptide at pH 6 with iodoacetic anhydride selectively modifies the N-terminus by exploiting the pK(a) differences between the alpha-amino group and any lysine side-chain epsilon-amino groups. The iodoacetyl peptide can react with many reagents to form a fixed positive charge. We find reaction with dimethyloctylamine forms a quaternary ammonium derivative with good surface activity properties and concomitant increased sensitivity. The high-energy CAD fragment ion spectra of the N-terminally derivatized peptides show predominantly a(n) and d(n) ions. The abundant d(n) ions permit ready distinction of leucine and isoleucine. Fewer fragment ions make data interpretation simpler and lead to more intense peaks since the ion intensity is spread among fewer peaks. The method is particularly useful for peptides which do not otherwise yield sufficient fragmentation to provide either the complete sequence or the locations of modified amino acids.
Stults
J T
JT
Protein Chemistry Department, Genentech, Inc., South San Francisco, California 94080-4990.
Lai
J
J
McCune
S
S
Wetzel
R
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D013006:Growth Hormone / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D013947:Thymosin / Q000737:chemistry
D000596:Amino Acids; D010446:Peptide Fragments; D013947:Thymosin; D013006:Growth Hormone