Optimization of titanium dioxide and immunoaffinity-based enrichment procedures for tyrosine phosphopeptide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

Tyrosine phosphorylation is an important regulator of signaling in cellular pathways, and dysregulated tyrosine phosphorylation causes several diseases. Mass spectrometry has revealed the importance of global phosphoproteomic characterization. Analysis of tyrosine phosphorylation by studying the mass-spectrometry (MS)-determined phosphoproteome remains difficult because of the relatively low abundance of tyrosine phosphoproteins. To effectively evaluate tyrosine-phosphopeptide enrichment and reduce ion suppression from non-phosphorylated peptides in MS analysis, three trypsin-digested BSA peptides and 14 standard phosphopeptides, including six tyrosine phosphopeptides, four serine phosphopeptides, and four threonine phosphopeptides, were subjected to titanium dioxide immunoaffinity-based enrichment and also to combined enrichment using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-mass spectrometry (LC-MS) analyses. The enrichment factors were evaluated to determine the efficiency of each enrichment procedure. Comparison of five optimized enrichment methods, including TiO2-based immunoaffinity purification in Tris and MOPS buffer systems, TiO2-immunoaffinity enrichment, and immunoaffinity-TiO2 enrichment for total tyrosine, serine and threonine phosphopeptides, revealed that the order of the enrichment factors for total tyrosine phosphopeptides is: (i) immunoaffinity-TiO2 (enrichment factor = 38,244), (ii) TiO2-immunoaffinity (enrichment factor = 24,987), (iii) TiO2 micro-column (enrichment factor = 10,305), (iv) immunoaffinity in Tris buffer system (enrichment factor = 1450), and (v) immunoaffinity in the MOPS buffer system (enrichment factor = 32). These results reveal that an alternative enrichment scheme before use of a TiO2 micro-column, using immunoaffinity 4G10 and PY99 antibody enrichment under optimized conditions, can provide greater selectivity for tyrosine-phosphopeptide enrichment.

Identification of Phosphorylated Cyclin-Dependent Kinase 1 Associated with Colorectal Cancer Survival Using Label-Free Quantitative Analyses

“…After the LC-MS analysis, the results showed that 20,000 monoisotopic peaks were present in each LC-MS analysis. To produce a high yield of phosphopeptide signals for further targeted LC-MS/MS analysis in the iPhos analysis [ 32 ], the iPhos analytical parameters were set as follows, a maximum number of 1000 for the LC-MS dataset of phosphorylation and dephosphorylation pairs; a mass tolerance of 0.1 Da; a retention time tolerance after dephosphorylation within five min [ 17 , 20 , 33 ]; and a charge state of ≧2. The signals selected from the comparison with the signals for phosphorylation and dephosphorylation by iPhos showed that a total of 365 m / z signals were filtered from 6 LC-MS analyses of phosphorylation and dephos [ 17 , 34 ]phorylation pairs for prepared SW480 and SW620 cells ( Fig 2A ).…”

Enrichment Strategies for Identification and Characterization of Phosphoproteome

“…TiO 2 techniques are usually performed using self-packed or commercial products with microcolumn, 17,56 or beads. 20,25,40 To improve the reproducibility of TiO 2 for phosphopeptide enrichment, Tape and co-worker described that the automatic phosphopeptide enrichment using magnetic TiO 2 beads with IMAC.…”

“…59 In many reports, acidic loading buffers on TiO 2 have been tested and optimized with the modifiers including 2, 5-dihydroxybenzoic acid (DHB), malic acid, lactic acid, and other acidic reagents to enhance the enrichment efficiency of phosphopeptide. [17][18][19][20][21] Capacity of acidic modifier in loading buffer for removing non-specific binding decreased as follows: DHB = salicylic acid = phthalic acid > benzoic acid = cyclohexanecarboxylic acid > phosphoric acid > TFA > acetic acid. 53 In recently, specificity of TiO 2 chromatography was optimized by adding glycerol 60 or citric acid.…”

“…A variety of enrichment and fractionation methods in phosphoproteomics have been reported including the enrichment with immobilized metal affinity chromatography (IMAC) 6,[14][15][16] and metal oxide affinity chromatography (MOAC) as well as titanium dioxide (TiO 2 ), [17][18][19][20][21][22][23][24][25] and fractionation with strong cation exchange (SCX), 26,27 hydrophilic interaction chromatography (HILIC), [28][29][30] and electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). [31][32][33] More recently, the two sequential steps of preparation methods are commonly used in large scale phosphoproteomics.…”

Selective adsorption of protein by a high-efficiency Cu2+-cooperated magnetic imprinted nanomaterial

“…Therefore, the depletion of high‐abundance proteins is prerequisite step in facilitating access to analysis low‐abundance proteins. To date, a multitude of methods have been used to capture the high‐abundance proteins from biological samples, including immune‐affinity chromatography , capillary electrophoresis (CE) , and immobilized metal affinity chromatography . However, low selectivity, time‐consuming process, poor stability, or high cost limit their practical application.…”