Methods for on-chip protein analysis.
The unambiguous identification of peptides/proteins is crucial for the definition of the proteome. Using ProteinChip Array technology also known as surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS), we developed experimental protocols and probed test conditions required for the protein identification on ProteinChip surfaces. We were able to directly digest peptides/proteins on-chip surfaces by specific proteases, such as trypsin, and to obtain the peptide mass fingerprint of the sample under investigation by its direct analysis on a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry was performed on several of the resulting tryptic peptides by using collision quadrupole time of flight (Qq-TOF) MS/MS via the ProteinChip interface, thus allowing the unambiguous identification of the protein(s) within the sample. In addition, we were able to identify the C-terminal sequence of peptides by their digestion with carboxypeptidase Y directly on ProteinChip surfaces coupled with SELDI-TOF MS analysis of the resulting peptide mass ladders employing the instrument's protein ladder sequence software. Moreover, the removal of up to nine amino acid residues from the C-terminal end of a peptide extends the functional range of Qq-TOF MS/MS sequence determination to over 3000 m/z. The utility of these procedures for the proteome exploration are discussed.
Caputo
Emilia
E
Unit on Molecular Structures, LNT, NIMH, NIH, DHHS, 10 Center Drive, Bldg. 10 3N309, Bethesda, MD 20892-1262, USA.
Moharram
Ramy
R
Martin
Brian M
BM
D000595:Amino Acid Sequence; D043402:Cathepsin A / Q000378:metabolism; D030562:Databases, Protein; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D011506:Proteins; D043402:Cathepsin A; D014357:Trypsin