Electrophoretic mobility for peptides with post-translational modifications in capillary electrophoresis.
Several semiempirical models for peptide electrophoretic mobility have been tested for capillary electrophoresis (CE) separation with a positively charged capillary using on-line CE combined with electrospray ionization-mass spectrometry (ESI-MS). In the current system with pH 2.7, the expression q/M(0.56) provided the best correlation with the electrophoretic mobility in the analysis of a set of 18 standard samples, where q is the calculated net charge and M is the molecular weight. The peptides resulting from various digests of horse heart myoglobin or bovine hemoglobin were used to demonstrate the validity of this correlation. Post-translationally modified peptides from tryptic digest of human myelin basic protein were also investigated and were found to provide excellent correlation with the linear plot when the total charge of the peptide was correctly calculated. If the total charge was not properly calculated then the post-translationally modified peptides fell off the linear plot. Using this method five arginine residues (residues 5, 49, 54, 97 and 130) were found to be partially citrullinated, four glutamine residues (residues 8, 103, 121 and 147) were found to be partially deamidated and both methionines at residues 21 and 167 were found to be partially oxidized. Three peptides were found with phosphorylation; TPPPSQGK (residue 98 to 105), FSWGAEGQR (residue 114 to 122), and SGSPMAR (residue 163 to 169) and Arginine at 107 was found to be partially monomethylated or dimethylated. The method may provide an excellent means of identifying the presence of peptides with post-translational modifications in conjunction with MS.
Kim
Jeongkwon
J
Department of Chemistry, University of Michigan, Ann Arbor 48109-1055, USA.
Zand
Robert
R
Lubman
David M
DM
D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin; D019075:Electrophoresis, Capillary / Q000379:methods*; D006454:Hemoglobins / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008433:Mathematics; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000032:analysis* / Q000737:chemistry; D009211:Myoglobin / Q000032:analysis; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin
D006454:Hemoglobins; D004676:Myelin Basic Protein; D009211:Myoglobin; D010446:Peptide Fragments; D010455:Peptides; D002918:Chymotrypsin; D014357:Trypsin