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.

Keywords: Gel fractionation; In-gel digestion; Method optimization; Quantification; Technology; iTRAQ.

Figure 1

The plotted relationship of the theoretical and experimental ratios (data in Table1) of two of the four ratios. Each data point represents the average of the log ratios of all MS/MS spectra. Error bars indicate the SD. The linear regression of the means results in an r² value of 0.9997 (114/115) and 0.9981 (116/117). The dashed lines define the 95% confidence band of the regression line.

Figure 2

(A) Left panel: Equal amounts of a HepG2 cell lysate are divided over 16 lanes of two gels, whereby a loaded lane is alternated with an empty one to allow easy cutting of the gel (schematic of one gel is shown). After electrophoresis, the gels are cut around the 50 kDa marker to create a high (A) and low (B) molecular weight fraction. The different lanes were excised and in-gel digestion was performed on the different gel bands according to different conditions and pooled for each replicate after labeling. Right panel: Condition 1—digestion of a fixed and SR-stained gel, marked as the standard (ST) protocol. Condition 2—digestion of a fixed, nonstained gel. Condition 3—digestion of a nonfixed, nonstained gel. Condition 4—ST supplemented with 1 mM CaCl₂ and 5% ACN. Extracted peptides from bands 1 to 4 were labeled and pooled according to the presented schedule, together forming four high and four low molecular weight samples. S1–S4: different replicates. (B) Each bar represents the average and SD of all the reporters of one of the four replicas. The different conditions are compared to the standard situation where the gel is fixed and SR stained. Despite a large variation, ratios indicate that SR has a negative effect on peptide recovery and fixation a positive influence. Addition of CaCl₂ and ACN during trypsin digestion increases the peptide yield over sevenfold in average. Asterisk: The positive effect of gel fixation is verified by three of four replicates from the “fixation no SR/no fixation no SR” ratios.