Identification of proteins by combination of size-exclusion chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and comparison of some desalting procedures for both intact proteins and their tryptic digests.
Separation of a protein mixture by size-exclusion chromatography (SEC) was combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Identification of proteins in the collected fractions was performed both as intact proteins by MALDI-TOFMS and using peptide mass fingerprinting (PMF) after their digestion with trypsin. The presence of salts mostly disturbs the MALDI-TOFMS signal and, therefore, proper purification or desalting procedures must be employed. Four desalting procedures (desalting column packed with Sephadex G-100, on-target washing, centrifugal filter devices and ZipTip C(18)) for purification of fractions of proteins separated by SEC and their tryptic digests prior to determination of their exact molecular masses by MALDI-TOFMS were compared. In the case of intact proteins, the experiments showed that the best desalting procedures are the use of ZipTip C(18) pipette tips and Ultrafree CL centrifugal filter devices. The peptide digests can be purified by using ZipTip C(18) pipette tips or on-target washing when both of these procedures provide similar results. On-target washing can be used as a simple procedure to improve the mass spectra of salt-containing samples. Analyses of the droplets collected after the on-target washing show losses of sample and matrix caused by dissolution of these compounds during this procedure. Further, it was found that protein identification based on PMF is more sensitive than analyses of intact proteins and that multiple on-target washing is very advantageous for analyses of peptide mixtures with a high content of salts.
Salplachta
Jirí
J
Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veverí 97, 611 42 Brno, Czech Republic.
Rehulka
Pavel
P
Chmelík
Josef
J
D002850:Chromatography, Gel / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin
D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin