Perhaps the greatest power of 2DE and related IEF techniques is the complementarity with mass spectrometry. When used together, this approach is able to reduce proteome complexity with enough resolution that single proteoforms can be analyzed with far more depth compared to a standard shotgun approach.
Figure 3 depicts several possible experimental IEF methodologies coupled with mass spectrometry. While the image obtained from the gel-based experiment provides information about the protein composition, complete determination of the protein in the absence of a standard is challenging. It is now common practice to perform 2DE in order to separate proteins and visualize spots while also obtaining quantitative information, followed by excision of the spot and downstream mass spectrometry analysis. Mass spectrometry plays a crucial role in analyte identification and characterization, particularly for peptide and protein analysis with the advancement in soft ionization techniques (e.g., matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI)).
Although there have been significant advances in regards to proteomic analysis via mass spectrometry, undersampling of the proteome and consequently the inability to obtain deep proteome coverage remains a problem when analyzing complex samples. For this reason, the use of multidimensional separation methods including IEF have been employed in order to reduce sample complexity prior to analysis. With the development of multidimensional protein identification technology (MudPIT, also termed shotgun), in which two-dimensional chromatography was carried out, extensive proteome coverage was obtained [
74]. Following this development, a variety of alternative approaches were reported, many including the use of pI-based separations to fractionate prior to mass spectrometry analysis [
75]. There are numerous reports in the literature that compare different separation methods that encompass IEF prior to tandem mass spectrometry. One of the most common orthogonal approaches used to resolve highly complex proteomes is the use of IEF-IPG and reverse phase liquid chromatography [
76]. Another study showed that a three-dimensional IEF based approach provided superior resolution compared to previously reported 2D-gel electrophoresis results for the discovery of biomarkers when exploring the microbial metaproteomes in plants [
77]. Interestingly, the use of high-pH reverse phase fractionation prior to mass spectrometry analysis has shown to result in a more uniform distribution of unique peptides across all fractions collected compared to off-gel IEF [
78]. It is important to note, however, that interfering agents, found in common isobaric labeling kits (e.g., iTRAQ, Sciex, Framingham, MA, USA and TMT, TheroFisher Scientific, Waltham, MA, USA) cannot be removed via this method. Lengqvist and colleagues demonstrated that not only is IPG-IEF compatible with labeling techniques such as iTRAQ, but this separation method is also able to provide additional experimental data such as peptide pI in addition to high resolution fractionation [
79]. Many of the above-mentioned techniques have been used in this approach for high resolution fractionation, protein identification and quantification by mass spectrometry.