Identification of low molecular weight proteins isolated by 2-D liquid separations

• Published in: Journal of mass spectrometry. Vol. 39; no. 7; pp. 770 - 780
• Main Authors: Zhu, Kan, Miller, Fred R., Barder, Timothy J., Lubman, David M.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.07.2004; Wiley
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; breast cancer; Breast Neoplasms - chemistry; Cell Line, Tumor - chemistry; Chemistry; Chromatography, High Pressure Liquid - methods; Exact sciences and technology; Female; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Humans; liquid separation; low molecular weight protein; Mass spectrometry; matrix-assisted laser desorption/ionization; Molecular Weight; Organic chemistry; peptide identification; Proteins; Proteins - chemistry; Proteins - isolation & purification; quadrupole time-of-flight; Reactivity and mechanisms; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; two-dimensional; quadrupole time-of-flight; Chemical analysis; Peptides; low molecular weight protein; HPLC chromatography; breast cancer; liquid separation; Matrix assisted laser desorption ionization; Protein; Reversed phase chromatography; two-dimensional; Peptide fragment; Time of flight method; matrix-assisted laser desorption/ionization; Low molecular weight compound; peptide identification; Qualitative analysis; Mass spectrometry
• ISSN: 1076-5174; 1096-9888
• DOI: 10.1002/jms.650

Proteins with molecular mass (Mr) <20 kDa are often poorly separated in 2‐D sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, low‐Mr 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 Mr 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 Mr 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. Copyright © 2004 John Wiley & Sons, Ltd.