1. Introduction

The first aim in human proteomics, as it was proposed by the Human Proteome Organization (HUPO), is a complete catalog of all human proteins. Due to collaborative efforts inside the Chromosome-based Human Proteome Project (C-HPP), this task is close to completion now [1, 2]. According to NextProt release from Aug 1, 2017, 17,168 from 20,199 predicted proteins have already been found. Still, ~3000 proteins are in the list of so-called "missing proteins." But even after completion of this task, only representative proteins will be identified in most cases [3, 4]. The situation here is much more complicated, as proteins can exist as different proteoforms (protein species) [5–7]. Proteoforms, as the smallest units of the proteome, are molecules (polypeptides) arising from all combinatorial sources of variation after

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expression of a single gene. Each proteoform is a chemically clearly defined molecule. These molecules are different due to genetic variation, alternatively spliced RNA, transcripts and posttranslational modifications [5, 7]. Accordingly, the term protein refers to its coding gene and, therefore, becomes as the umbrella term for all developing proteoforms/protein species [6]. Sometimes the term "proteoform" is used for the description of structural variants of proteins as well [8]. But it will make the issue of terminology in proteomics more complicated and confused, as even inside the abovementioned definition, a proteomics field of proteoforms is very broad and could encompass many billions of components [9–12]. For instance, all combinations of 30 known modifications of histone H3 alone can theoretically produce more than 1 billion of proteoforms [10, 13]. Because of such a variety, huge range of concentration (7–8 orders of magnitude in blood plasma), and dynamic changes during life cycle, their identification, quantitation and database organization is a serious challenge. Nevertheless, there is evident progress in this area. So far, the main workhorse in proteomics was bottom-up mass spectrometry, but the top-down approach is becoming pre-eminent today [14]. Top-down proteomics implies that mass spectrometry is applied at the proteoform level, allowing the acquisition of information about all intramolecular complexity preserved during analysis, that might be overlooked in bottom-up shotgun workflows [14, 15]. But top-down proteomics cannot be just a one-step procedure. There are also several approaches based on protein separation that are involved in proteoform analysis. Among these methods, two-dimensional gel electrophoresis (2DE) occupies the special place. Accordingly, different schemes could be used to establish a basis for a comprehensive knowledge base for protein/proteoform inventory.
