TY - JOUR
T1 - Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications
AU - Sugiyama, Naoyuki
AU - Masuda, Takeshi
AU - Shinoda, Kosaku
AU - Nakamura, Akihiro
AU - Tomita, Masaru
AU - Ishihama, Yasushi
PY - 2007/6
Y1 - 2007/6
N2 - 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 β-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 β-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.
AB - 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 β-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 β-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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U2 - 10.1074/mcp.T600060-MCP200
DO - 10.1074/mcp.T600060-MCP200
M3 - Article
C2 - 17322306
AN - SCOPUS:34347403192
SN - 1535-9476
VL - 6
SP - 1103
EP - 1109
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
IS - 6
ER -