Matrix-assisted laser desorption/ionization- quadrupole ion trap-time of flight mass spectrometry sequencing resolves structures of unidentified peptides obtained by in-gel tryptic digestion of haptoglobin derivatives from human plasma proteomes.
Two-dimensional gel electrophoresis-separated and excised haptoglobin alpha2-chain protein spots were subjected to in-gel digestion with trypsin. Previously unassigned peptide ion signals observed in mass spectrometric fingerprinting experiments were sequenced using the matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectrometer and showed that the haptoglobin alpha-chain derivative under study was cleaved by trypsin unspecifically. Abundant cleavages occurred C-terminal to histidine residues at H23, H28, and H87. In addition, mild acidic hydrolysis leading to cleavage after aspartic acid residues at D13 was observed. The uninterpreted tandem mass spectrometry (MS/MS) spectrum of the peptide with ion signal at 2620.19 was submitted to database search and yielded the identification of the corresponding peptide sequence comprising amino acids (aa) aa65-87 from the haptoglobin alpha-chain protein. Also, the presence of a mixture of two tryptic peptides (mass to charge ratio m/z 1708.8; aa40-54, and aa99-113, respectively), that is caused by a tiny sequence variation between the two repeats in the haptoglobin alpha2-chain protein was resolved by MS/MS fragmentation using the MALDI-QIT-TOF mass spectrometer instrument. Advantageous features such as (i) easy parent ion creation, (ii) minimal sample consumption, and (iii) real collision induced dissociation conditions, were combined successfully to determine the amino acid sequences of the previously unassigned peptides. Hence, the novel mass spectrometric sequencing method applied here has proven effective for identification of distinct molecular protein structures.
Koy
Cornelia
C
Proteome Center Rostock, University of Rostock, Rostock, Germany.
Mikkat
Stefan
S
Raptakis
Emmanuel
E
Sutton
Chris
C
Resch
Martin
M
Tanaka
Koichi
K
Glocker
Michael O
MO
D000483:Alleles; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry*; D014644:Genetic Variation; D006242:Haptoglobins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry*; D020543:Proteome; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001798:Blood Proteins; D006242:Haptoglobins; D010446:Peptide Fragments; D010455:Peptides; D020543:Proteome