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Comparison of metal and metal oxide media for phosphopeptide enrichment prior to mass spectrometric analyses.

ABSTRACT: Several affinity resins consisting of ionic metals or metal oxides were investigated for their phosphopeptide enrichment capabilities with subsequent mass spectrometric analyses. Commercially-available enrichment metal oxide affinity chromatography (MOAC) resins using manufacturer's and/or published protocols were compared and evaluated for the most efficient and selective method that could be implemented as a standard enrichment procedure. From these comparative analyses, using a tryptic digest of casein proteins, it was determined that in our hands, two of the resins out-performed the others based on a variety of criteria, including the number of phosphorylation sites identified during MS analyses, the lower numbers of nonspecifically bound peptides observed, and the limits of detection. Applicability of these enrichment resins to a complex biological mixture was investigated. For this work, a mixture of avian histones was digested, subjected to titanium dioxide phosphopeptide enrichment, and analyzed by mass spectrometry. Eight phosphorylated tryptic peptides were observed following enrichment and subsequent LC/MS/MS analyses. Of note, seven of the eight phosphopeptides were not observed without titanium dioxide enrichment. From these analyses, four sites of phosphorylation were unequivocally determined, two of which have not been reported previously. Four additional phosphopeptides were observed; however, the site of phosphorylation could not be distinguished but was localized to one of two possible amino acids. These methods should aid in the investigation of proteins post-translationally modified with phosphate, especially those present at low concentrations as was demonstrated by successful enrichment at the femtomole level.

SUBMITTER: Gates MB

PROVIDER: S-EPMC2982680 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Comparison of metal and metal oxide media for phosphopeptide enrichment prior to mass spectrometric analyses.

Gates Matthew B MB Tomer Kenneth B KB Deterding Leesa J LJ

Journal of the American Society for Mass Spectrometry 20100619 10

Several affinity resins consisting of ionic metals or metal oxides were investigated for their phosphopeptide enrichment capabilities with subsequent mass spectrometric analyses. Commercially-available enrichment metal oxide affinity chromatography (MOAC) resins using manufacturer's and/or published protocols were compared and evaluated for the most efficient and selective method that could be implemented as a standard enrichment procedure. From these comparative analyses, using a tryptic digest ...[more]

PMID: 20634090

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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 ?g 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:RationaleThe low molecular weight (LMW) proteins present in circulating body fluids, such as serum and plasma, hold biological significance as possible biomarkers. A major obstacle in mass spectrometry-based proteomics of serum is the presence of abundant high molecular weight proteins which mask the identification and quantitation of lower molecular weight proteins. Traditional methods involve the use of affinity resins to remove high molecular weight proteins, such as albumin and immunoglobulin G, with concomitant loss of lower molecular weight proteins. Considering the importance of depleting high molecular proteins, this paper compares an affinity resin, a gel-filter, and an acetonitrile (ACN) precipitation method to achieve successful removal of high molecular weight proteins and recovery of lower molecular weight proteins.MethodsSerum enrichment was carried out by multiple methods such as with the commercially available serum protein mini kit, ACN precipitation, and a gel filter method. Mass spectrometric runs were carried out on an AB SCIEX ESI QTOF 5600 mass spectrometer. Mass spectrometry analysis of the enriched serum obtained by ACN precipitation and gel filter method was performed for global proteome profiling. Quantitative mass spectrometry using isobaric tags for relative and absolute quantitation (iTRAQ) for ACN-precipitated enriched serum was also carried out.ResultsThe gel filter method, though allowing for the resolution and identification of LMW proteins, was better suited for global proteome analysis and not preferred for quantitative proteomic experiments. In contrast, enrichment by the ACN precipitation method allowed for the reproducible identification and quantitation of LMW proteins having molecular weight ≥4 kDa.ConclusionsUsing only chilled ACN and centrifugation, most of the highly abundant proteins were successfully removed from the serum, while recovering a significant portion of the LMW proteome. A more rapid protocol, which is compatible with iTRAQ labeling, to achieve improved results has been elucidated, thus allowing for better screening and identification of potential biomarkers.

Dataset Information

Comparison of metal and metal oxide media for phosphopeptide enrichment prior to mass spectrometric analyses.

Publications

Comparison of metal and metal oxide media for phosphopeptide enrichment prior to mass spectrometric analyses.

Similar Datasets

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