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

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 in...

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Published inJournal 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
LanguageEnglish
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
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Summary: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
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
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ISSN:1535-3893
1535-3907
DOI:10.1021/pr0604460