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Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides.

ABSTRACT: We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. Although our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semiquantitative method when used in large-scale phosphoproteomics studies in complex backgrounds.

SUBMITTER: Sun Z

PROVIDER: S-EPMC3909987 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides.

Sun Zeyu Z Hamilton Karyn L KL Reardon Kenneth F KF

Analytical biochemistry 20131001

We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptid ...[more]

PMID: 24096195

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Project description:Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO(2), Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 μg to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO(2) in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems.

Project description:Abstract: Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, methodologies to enrich the heterogeneous types of phosphopeptides are critical towards comprehensive mapping of the under-explored phosphoproteome. Taking advantage of the distinctive binding affinity of Ga3+ and Fe3+ towards phosphopeptides, we designed a tip-based metal-directed immobilized metal ion affinity (MD-IMAC) chromatography for sequential enrichment of phosphopeptides. On the analysis of Raji B cell, this sequential Ga3+-Fe3+-IMAC strategy demonstrated 1.5-3.5 fold superior phosphoproteomic coverage compared to the single IMAC (Fe3+, Ti4+, Ga3+ and Al3+). In addition, as high as 92% among the 6283 phosphopeptides was uniquely enriched by either 1st Ga3+-IMAC fraction (41%) or 2nd Fe3+-IMAC fraction (51%). The complementary property of Ga3+ and Fe3+ was further shown on the exclusively superior efficiency to enriched almost all the 1214 multiply phosphorylated peptides (99.4%) by 1st Ga3+-IMAC, while as low as 10% of 5069 monophosphorylated phosphopeptides was commonly enriched by both fractions. Application of our sequential Ga3+-Fe3+-IMAC approach to a human lung cancer tissue allowed the identification of 2560 unique phosphopeptides with only 8% overlapping. The fractionation ability was shown not only on the mono phosphopeptides and multiply phosphopeptides but also on the basic and acidic phosphopeptides; acidiphilic phosphorylation sites were predominately present in 1st Ga3+-IMAC (72%) and 85% Pro-directed and 79% basophilic phosphorylation sites were enriched by 2nd Fe3+-IMAC. Most interestingly, this strategy complementarily mapped different kinase substrate on the protein as well as site levels in multiple cellular pathways related to cancer invasion and metastasis of lung cancer ., Given the demonstrated fractionation ability, reproducibility, sensitivity and ease of tip preparation, we hope that this Ga3+-Fe3+-IMAC allow more comprehensive characterization of phosphoproteome in vitro and in vivo. Database search: The raw MS/MS data obtained by TripleTOF 5600 were processed using AB_SCIEX MS Data Converter with default parameters. All MS/MS files were analyzed using Mascot (Matrix Science, London, UK; version 2.3) against the SwissPort database (version 57.8) with the following constraints: an allowance for tryptic peptides of up to two missed cleavage sites, a fragment ion mass tolerance of 0.05 Da, and a parent ion tolerance of 10 ppm. Phosphorylation (S, T, Y) and oxidation (M) were selected as variable modifications. Searching on a randomized decoy database created by Mascot was required to evaluate the false discovery rate associated with protein identification. The false discovery rates with a Mascot score (p< 0.05) ranged between 0% and 1% in this study.

Project description:Magnetic materials in sample preparation for shotgun phosphoproteomics offer several advantages over conventional systems, as the enrichment can be achieved directly in solution, but they still suffer from some drawbacks, due to limited stability and selectivity, which is supposed to be affected by the hydrophilicity of the polymeric supports used for cation immobilization. The paper describes the development of an improved magnetic material with increased stability, thanks to a two-step covering of the magnetic core, for the enrichment of phosphopeptides in biological samples. Four materials were prepared featuring a polymeric shell with tunable hydrophilicity, obtained by "grafting from" polymerization of glycidyl methacrylate with 0-8.3% of polyethylene glycol methacrylate (PEGMA), the latter used to modulate the hydrophilicity of the material surface. Finally, the materials were functionalized with iminodiacetic acid for Ti4+ ion immobilization. The materials were analyzed for their composition by a combination of CHN elemental analysis and thermogravimetric analysis, also hyphenated to gas chromatography and mass spectrometric detection. Surface characteristics were evaluated by water contact angle measurements, scanning electron microscopy and energy dispersive X-ray spectrometry. These materials were applied to the enrichment of phosphopeptides from yeast protein digests. Peptides were identified by proteomics techniques using nano-high performance liquid chromatography coupled to mass spectrometry and bioinformatics. Qualitatively the peptides identified by the four systems were comparable, with 1606-1693 phosphopeptide identifications and a selectivity of 47-54% for all materials. The physico-chemical features of the identified peptides were also the same for the four materials. In particular, the grand average of hydropathy index values indicated that the enriched phosphopeptides were hydrophilic (ca. 90%), and only some co-enriched non-phosphorylated peptides were hydrophobic (21-28%), regardless of the material used for enrichment. Peptides had a pI???7, which indicated a well-known bias for acidic peptides binding, attributed to the interaction with the metal center itself. The results indicated that the enrichment of phosphopeptides and the co-enrichment of non-phosphorylated peptides is mainly driven by interactions with Ti4+ and does not depend on the amount of PEGMA chains in the polymer shell.

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Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides.

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Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides.

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