Abstract
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.
Highlights
We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC)
We developed new approaches for phosphopeptide enrichment using MOC modified with aliphatic hydroxy acids
Effect of Hydroxy Acids on Phosphopeptide Enrichment—A digested phosphoprotein standard mixture of ␣-casein, fetuin, and phosvitin was used to evaluate the effect of hydroxy acids in the loading buffer on selective enrichment of phosphopeptides with titania, zirconia, alumina, Al(OH)31⁄7xH2O, and boehmite MOC tips (Table I)
Summary
Materials—Titania (titanium dioxide; particle size, 10 m) was obtained from GL Sciences (Tokyo, Japan). Digestion of Standard Phosphoproteins—␣-Casein, fetuin, and phosvitin were individually reduced with DTT, alkylated with iodoacetamide, and digested with Lys-C followed by dilution and trypsin digestion as described previously [28]. These digested samples were desalted using StageTips with C18 Empore disk membranes [29]. Peptides and proteins were identified by means of automated database searching using Mascot version 2.1 (Matrix Science, London, UK) against UniProt/Swiss-Prot release 9.0 (October 31, 2006) with a precursor mass tolerance of 50 ppm (QSTAR) or 3 ppm (LTQ-Orbitrap), a fragment ion mass tolerance of 0.25 Da (QSTAR) or 0.8 Da (LTQ-Orbitrap), taxonomy of human, and strict trypsin specificity [33] allowing for up to two missed cleavages.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
