Ti4+-phosphate functionalized cellulose for phosphopeptides enrichment and its application in rice phosphoproteome analysis

In this study, a novel immobilized metal ion affinity chromatography (IMAC) material for phosphopeptide enrichment was prepared based on modified cellulose and was applied in rice phosphoproteome analysis. Firstly, cellulose was modified with phosphoric acid via esterification, and then Ti4+ was chelated onto the phosphorylated cellulose. The synthesized materials were ultrafine powders and had good dispersibility in acidic buffer, and as supporting matrix, phosphorylated cellulose exhibited good biocompatibility and chemical stability. Enrichment conditions were optimized and the optimum loading buffer was 40% acetonitrile (ACN) with 6% trifluoroacetic acid (TFA). Finally, the Ti4+-phosphate functionalized cellulose was submitted to phosphopeptides enrichment prior to mass spectrometry (MS). For α-casein lysates, 14 phosphopeptides were detected with high intensities even though the sample concentration was as low as 2 pmol. Besides, 15 phosphopeptides were still identified by using the digest mixture of α-casein and bovine serum albumin with molar ratio of 1:100, which demonstrated high specificity and sensitivity for phosphopeptides enrichment. 19 phosphoproteins were identified from 200 μg of salt-free rice leaf protein lysates, while 30 phosphoproteins were identified from salt-stressed rice leaf protein lysates, and most of these proteins were related to the biological processes in response to abiotic stimulus.