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Molecular & Cellular Proteomics 6:1103-1109, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

Phosphopeptide Enrichment by Aliphatic Hydroxy Acid-modified Metal Oxide Chromatography for Nano-LC-MS/MS in Proteomics Applications^*,S

Naoyuki Sugiyama^,, Takeshi Masuda, Kosaku Shinoda^,, Akihiro Nakamura, Masaru Tomita^, and Yasushi Ishihama^,¶,||

From the Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan, Human Metabolome Technologies, Kakuganji, Tsuruoka 997-0052, Japan, and ^¶ Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and ß-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and ß-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates.

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