Construction of 2DE Patterns of Plasma Proteins: Aspect of Potential Tumor Markers

Abstract

The use of tumor markers aids in the early detection of cancer recurrence and prognosis. There is a hope that they might also be useful in screening tests for the early detection of cancer. Here, the question of finding ideal tumor markers, which should be sensitive, specific, and reliable, is an acute issue. Human plasma is one of the most popular samples as it is commonly collected in the clinic and provides noninvasive, rapid analysis for any type of disease including cancer. Many efforts have been applied in searching for “ideal” tumor markers, digging very deep into plasma proteomes. The situation in this area can be improved in two ways—by attempting to find an ideal single tumor marker or by generating panels of different markers. In both cases, proteomics certainly plays a major role. There is a line of evidence that the most abundant, so-called “classical plasma proteins”, may be used to generate a tumor biomarker profile. To be comprehensive these profiles should have information not only about protein levels but also proteoform distribution for each protein. Initially, the profile of these proteins in norm should be generated. In our work, we collected bibliographic information about the connection of cancers with levels of “classical plasma proteins”. Additionally, we presented the proteoform profiles (2DE patterns) of these proteins in norm generated by two-dimensional electrophoresis with mass spectrometry and immunodetection. As a next step, similar profiles representing protein perturbations in plasma produced in the case of different cancers will be generated. Additionally, based on this information, different test systems can be developed.

Optimized Proteome Reduction for Integrative Top–Down Proteomics

“…Although there have been previous reports assessing, to some extent, the use of reducing reagents in 2DE, there does not appear to be a direct assessment between different sample types treated with these reagents, either alone or in combination [ 27 , 35 , 37 , 38 , 46 , 51 , 52 ]. It is acknowledged that there will not be a ‘perfect’ one-size-fits-all method; however, the goal here was to establish an optimized general protocol to enhance 2DE resolution and thus a breadth of research supported by routine integrative top–down proteomic analyses [ 7 , 8 , 9 , 11 , 12 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 ]. The data clearly indicate that for native proteome extracts, reduction using 100 mM DTT + 5 mM TBP prior to sample rehydration into IPG strips is superior to the current ‘standard’ as well as to other reagents previously reported in the literature.…”

“…However, despite its popularity, an appropriate, effective concentration to ensure a complete reduction of proteoforms in native proteomes, particularly during IEF, appeared not to have previously been quantitatively established. As with many routine aspects of 2DE, and indeed many analytical protocols, this was another which seemed somewhat ‘historically’ based on whichever earlier protocol was inherited or adopted from the literature; thus, concentrations generally in the range of 1–100 mM have been widely employed [ 7 , 8 , 19 , 24 , 62 , 63 , 64 , 65 ]. To determine an effective concentration, we first aimed to test for a relationship between disulfide bond content and DTT concentration using commercially purified protein standards.…”

“…To address the full complexity of proteomes, that is, routinely analyzing the constituent proteoforms beyond simply cataloguing canonical amino acid sequences, we must implement deep, quantitative analyses having the necessary analytical power and rigour [ 1 , 2 , 3 , 5 ]. Currently, this is best (and routinely) implemented using integrative top–down proteomic analyses that fully utilize the complementary strengths of 2DE and LC/TMS [ 1 , 2 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 18 , 19 , 22 , 69 ]. In contrast to bottom–up analyses, which infer the identity of only canonical protein sequences or mass-spectrometry-intensive top–down approaches that, while sensitive and thorough, can generally only handle species of <20–30 kDA, integrative top–down analyses using 2DE as the front-end of resolution enable deep proteomic analyses at the necessary level of proteoforms.…”

“…In contrast to bottom–up analyses, which infer the identity of only canonical protein sequences or mass-spectrometry-intensive top–down approaches that, while sensitive and thorough, can generally only handle species of <20–30 kDA, integrative top–down analyses using 2DE as the front-end of resolution enable deep proteomic analyses at the necessary level of proteoforms. The data presented in this study, along with decades of significant refinements and improvements, enable a genuine understanding of the complexity and diversity of the proteome by increasing analytical rigor [ 2 , 4 , 6 , 7 , 8 , 9 , 10 , 18 ].…”

“…Integrative top–down proteomics—tightly coupling two-dimensional gel electrophoresis (2DE; isoelectric focusing (IEF) followed by SDS-PAGE), liquid chromatography, and tandem mass spectrometry (LC/TMS), is currently the only available analytical approach that fully addresses the depth needed for effective and routine assessments of proteomes. In this regard, 2DE-based top–down proteomics has been widely employed for quantitative proteome analysis due to its well-established capacity to resolve many thousands of intact proteoforms from complex native proteome extracts [ 4 , 5 , 6 , 7 , 8 , 9 , 10 ]. Since its introduction, 2DE methodology has undergone rigorous review and assessment to improve resolution, detection/sensitivity, and thus, overall analytical rigour [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ].…”