Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications.
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 beta-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 beta-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.
Sugiyama
Naoyuki
N
Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan.
Masuda
Takeshi
T
Shinoda
Kosaku
K
Nakamura
Akihiro
A
Tomita
Masaru
M
Ishihama
Yasushi
Y
D000595:Amino Acid Sequence; D002457:Cell Extracts / Q000737:chemistry; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D006880:Hydroxy Acids / Q000737:chemistry* / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results
D002457:Cell Extracts; D006880:Hydroxy Acids; D010748:Phosphopeptides