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.

Functional and Integrative Analysis of the Proteomic Profile of Radish Root under Pb Exposure

“…However, there were some limitations in its applications such as poor reproducibility, weak sensitivity and low automation (Sazuka et al, 2004). Isobaric tags for relative and absolute quantification (iTRAQ) is a robust mass spectrometry of protein quantitative technology, which can perform relative and absolute quantification in up to eight samples in parallel (Bindschedler and Cramer, 2011; Glibert et al, 2014). Recently, iTRAQ has been widely used for large-scale quantitative plant proteomic studies in exploration of various metabolic processes at the post-transcriptional level (Kambiranda et al, 2013; Martínez-Esteso et al, 2013) in response to various stresses (Yang et al, 2014; Li et al, 2015; Fu et al, 2016).…”

A year (2014–2015) of plants in Proteomics journal. Progress in wet and dry methodologies, moving from protein catalogs, and the view of classic plant biochemists

“…It is simple and does not require much manipulation of the sample. The iTRAQ, quite popular among the Chinese groups, appeared as the third alternative, even though plant proteomicists did not pay much attention in the past to labeling techniques . By using this technique, Pan et al.…”

“…It is simple and does not require much manipulation of the sample. The iTRAQ, quite popular among the Chinese groups, appeared as the third alternative, even though plant proteomicists did not pay much attention in the past to labeling techniques [52][53][54][55]. By using this technique, Pan et al [56] have reported up to 13 706 and 12 124 proteins identified with an LTQ-Orbitrap hybrid mass spectrometer in A. thaliana roots and leaves.…”

Quality control in mass spectrometry‐based proteomics

“…As there are often multiple ways to conduct a single step to optimize the sample preparation a careful performance evaluation of the alternative options is required. To this end isobaric labeling techniques, such as isobaric tags for relative and absolute quantitation (iTRAQ), can be employed . Using iTRAQ each experimental condition under investigation can be differentially labeled, multiplexed, and analyzed simultaneously, after which the relative yield of each condition can be determined based on the associated reporter ions.…”