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ITRAQ as a method for optimization: enhancing peptide recovery after gel fractionation.

ABSTRACT: At the dawn of a new era in label-free quantitation on high-resolution MS instruments, classical methods such as iTRAQ continue to provide very useful insights in comparative proteomics. The potential to multiplex samples makes this reporter-based labeling technique highly suited for method optimization as demonstrated here by a set of standard series. Instead of studying ratios of annotated proteins, we propose an alternative method, based on the analysis of the average reporter ratios of all the spectra from a sample or a large distinct subset herein. This strategy circumvents the bias, associated with the annotation and iTRAQ quantitation, leading to increased adequacy in measuring yield differences between workflows. As gel electrophoresis prior to MS analysis is highly beneficial, for example, as a fractionation step, the approach was applied to evaluate the influence of several parameters of the established in-gel digestion protocol. We quantified the negative effect of SYPRO Ruby staining and the positive effect of gel fixation prior to digestion on peptide yield. Finally, we emphasize the benefits of adding CaCl2 and ACN to a tryptic in-gel digest, resulting in an up to tenfold enhanced peptide recovery and fewer trypsin missed cleavages.

SUBMITTER: Glibert P

PROVIDER: S-EPMC4413792 | biostudies-other | 2014 Mar

REPOSITORIES: biostudies-other

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iTRAQ as a method for optimization: enhancing peptide recovery after gel fractionation.

Glibert Pieter P Van Steendam Katleen K Dhaenens Maarten M Deforce Dieter D

Proteomics 20140218 6

At the dawn of a new era in label-free quantitation on high-resolution MS instruments, classical methods such as iTRAQ continue to provide very useful insights in comparative proteomics. The potential to multiplex samples makes this reporter-based labeling technique highly suited for method optimization as demonstrated here by a set of standard series. Instead of studying ratios of annotated proteins, we propose an alternative method, based on the analysis of the average reporter ratios of all t ...[more]

PMID: 24449435

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Project description:The isotopic fractionation factor and element partition coefficient can be calculated only after the geometric optimization of the molecular clusters is completed. Optimization directly affects the accuracy of some parameters, such as the average bond length, molecular volume, harmonic vibrational frequency, and other thermodynamic parameters. Here, we used the improved volume variable cluster model (VVCM) method to optimize the molecular clusters of a typical oxide, quartz. We documented the average bond length and relative volume change. Finally, we extracted the harmonic vibrational frequencies and calculated the equilibrium fractionation factor of the silicon and oxygen isotopes. Given its performance in geometrical optimization and isotope fractionation factor calculation, we further applied the improved VVCM method to calculate isotope equilibrium fractionation factors of Cd and Zn between the hydroxide (Zn-Al layered double hydroxide), carbonate (cadmium-containing calcite) and their aqueous solutions under superficial conditions. We summarized a detailed procedure and used it to re-evaluate published theoretical results for cadmium-containing hydroxyapatite, emphasizing the relative volume change for all clusters and confirming the optimal point charge arrangement (PCA). The results showed that the average bond length and isotope fractionation factor are consistent with those published in previous studies, and the relative volume changes are considerably lower than the results calculated using the periodic boundary method. Specifically, the average Si-O bond length of quartz was 1.63 Å, and the relative volume change of quartz centered on silicon atoms was - 0.39%. The average Zn-O bond length in the Zn-Al-layered double hydroxide was 2.10 Å, with a relative volume change of 1.96%. Cadmium-containing calcite had an average Cd-O bond length of 2.28 Å, with a relative volume change of 0.45%. At 298 K, the equilibrium fractionation factors between quartz, Zn-Al-layered double hydroxide, cadmium-containing calcite, and their corresponding aqueous solutions were [Formula: see text], [Formula: see text], [Formula: see text] and [Formula: see text] respectively. These results strongly support the reliability of the improved VVCM method for geometric optimization of molecular clusters.

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ITRAQ as a method for optimization: enhancing peptide recovery after gel fractionation.

Publications

iTRAQ as a method for optimization: enhancing peptide recovery after gel fractionation.

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