Single-Step Inline Hydroxyapatite Enrichment Facilitates Identification and Quantitation of Phosphopeptides from Mass-Limited Proteomes with MudPIT
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- 作者：Bryan R. Fonslow ; Sherry M. Niessen ; Meha Singh ; Catherine C. L. Wong ; Tao Xu ; Paulo C. Carvalho ; Jeong Choi ; Sung Kyu Park ; John R. Yates ; III
- 刊名：Journal of Proteome Research
- 出版年：2012
- 出版时间：May 4, 2012
- 年：2012
- 卷：11
- 期：5
- 页码：2697-2709
- 全文大小：546K
- 年卷期：v.11,no.5(May 4, 2012)
- ISSN：1535-3907
文摘
Herein we report the characterization and optimization of single-step inline enrichment of phosphopeptides directly from small amounts of whole cell and tissue lysates (100鈥?00 渭g) using a hydroxyapatite (HAP) microcolumn and Multidimensional Protein Identification Technology (MudPIT). In comparison to a triplicate HILIC-IMAC phosphopeptide enrichment study, 80% of the phosphopeptides identified using HAP-MudPIT were unique. Similarly, analysis of the consensus phosphorylation motifs between the two enrichment methods illustrates the complementarity of calcium- and iron-based enrichment methods and the higher sensitivity and selectivity of HAP-MudPIT for acidic motifs. We demonstrate how the identification of more multiply phosphorylated peptides from HAP-MudPIT can be used to quantify phosphorylation cooperativity. Through optimization of HAP-MudPIT on a whole cell lysate we routinely achieved identification and quantification of ca. 1000 phosphopeptides from a 1 h enrichment and 12 h MudPIT analysis on small quantities of material. Finally, we applied this optimized method to identify phosphorylation sites from a mass-limited mouse brain region, the amygdala (200鈥?00 渭g), identifying up to 4000 phosphopeptides per run.