Rapid Enrichment of Phosphopeptides and Phosphoproteins from Complex Samples Using Magnetic Particles Coated with Alumina as the Concentrating Probes for MALDI MS Analysis

• Published in: Journal of proteome research Vol. 6; no. 1; pp. 316 - 325
• Main Authors: Chen, Chen, Wei-Yu, Tsai, Pei-Jane, Chien, Kun-Yi, Yu, Jau-Song, Chen, Yu-Chie
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.01.2007
• Subjects: Aluminum Oxide - chemistry; Amino Acid Sequence; Carcinoma, Squamous Cell - metabolism; Caseins - chemistry; Cell Line, Tumor; Humans; Magnetics; Molecular Sequence Data; Peptide Mapping; Phosphopeptides - chemistry; Phosphoproteins - chemistry; Protein Denaturation; Proteomics - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - instrumentation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Trypsin - chemistry
• DOI: 10.1021/pr0604460
• ISSN: 1535-3893

In this study, we used nanocomposite magnetic particles coated with alumina as the affinity probes to selectively concentrate phosphorylated peptides and proteins from a low volume of sample solution. Tryptic digest products of phosphoproteins including α- and β-caseins, human protein phosphatase inhibitor 1, nonfat milk, egg white, and a cell lysate were used as the samples to demonstrate the feasibility of this approach. In only 30 and 90 s, phosphopeptides and phosphoproteins sufficient for characterization by MALDI-MS were enriched by the particles, respectively. Proteins trapped on the particles could be directly digested on the particles. The same particles in the digest solution were employed for enrichment of phosphopeptides. We estimated the required time for performing the enrichment of phosphopeptides from complex samples and characterization by MALDI MS was within 5 min. A small volume (50 μL) and a low concentration (5 × 10-10 M) of tryptic digest product of a phosphoprotein sample could be dramatically enriched and characterized using this approach. Keywords: alumina • magnetic particles • phosphopeptides • phosphoproteins • MALDI MS