Identification of low molecular weight proteins isolated by 2-D liquid separations.
Proteins with molecular mass (M(r)) <20 kDa are often poorly separated in 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, low-M(r) proteins may not be readily identified using peptide mass fingerprinting (PMF) owing to the small number of peptides generated in tryptic digestion. In this work, we used a 2-D liquid separation method based on chromatofocusing and non-porous silica reversed-phase high-performance liquid chromatography to purify proteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis and protein identification. Several proteins were identified using the PMF method where the result was supported using an accurate M(r) value obtained from electrospray ionization TOFMS. However, many proteins were not identified owing to an insufficient number of peptides observed in the MALDI-TOF experiments. The small number of peptides detected in MALDI-TOFMS can result from internal fragmentation, the few arginines in its sequence and incomplete tryptic digestion. MALDI-QTOFMS/MS can be used to identify many of these proteins. The accurate experimental M(r) and pI confirm identification and aid in identifying post-translational modifications such as truncations and acetylations. In some cases, high-quality MS/MS data obtained from the MALDI-QTOF spectrometer overcome preferential cleavages and result in protein identification.
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Kan
K
Zhu
Fred R
FR
Miller
Timothy J
TJ
Barder
David M
DM
Lubman
D001943:Breast Neoplasms / Q000737:chemistry; D045744:Cell Line, Tumor / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D011506:Proteins