A database for inventory of proteoform profiles: “2DE‐pattern”

Abstract

The human proteome is composed of a diverse and heterogeneous range of gene products/proteoforms/protein species. Because of the growing amount of information about proteoforms generated by different methods, we need a convenient approach to make an inventory of the data. Here, we present a database of proteoforms that is based on information obtained by separation of proteoforms using 2DE followed by shotgun ESI–LC–MS/MS. The database's principles and structure are described. The database is called “2DE‐pattern” as it contains multiple isoform‐centric patterns of proteoforms separated according to 2DE principles. The database can be freely used at http://2de-pattern.pnpi.nrcki.ru.

Proteomes Are of Proteoforms: Embracing the Complexity

“…Most notable, however, being a mature technology, these issues have been addressed and the approach has been substantially refined over the last two or more decades to address purported shortcomings ( Section 4.2.2 ). Unfortunately, many of the negative claims that still appear in review articles have simply become dogma, often perpetuated by those who have no experience with the technique, nor certainly reviewed the relevant primary literature from at least the last twenty years [ 1 , 29 , 49 , 81 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 ]. Simply, refined 2DE and its modifications can effectively resolve many (hundreds of) thousands of proteoforms across a broad range of classes and physico-chemical characteristics (i.e., soluble, membrane, acidic, basic, large, and small) including those of low abundance, and do so in parallel technical replicates [ 27 , 29 , 81 ].…”

“…The importance of the two separate dimensions lies in the fact that canonical proteins speciate into proteoforms that differ in MW and/or p I , and thus resolve into different spots on the gel, despite having identical (i.e., canonical) amino acid backbones ( Figure 6 ). Following staining and quantitative image analysis, select protein spots are excised from the gel and proteolytically digested prior to LC/MS/MS [ 81 , 98 , 102 , 104 , 105 , 110 , 111 , 112 ]. Selective staining, deep imaging, and third-dimension separations (3DE) may also be employed to further improve resolution ( Section 4.2.2 ).…”

“…These spots, along with areas at the pH extremes and unresolved small species/peptides in the migrating front, can be subjected to a third round of electrophoretic separations (i.e., 3DE), further enhancing the depth of proteome analysis [ 96 , 119 ]. While it was originally thought that one spot on a 2D gel contained perhaps 1–2 protein species, deeper analyses with ever more sensitive mass spectrometers has confirmed that there can be in the range of 200 or more proteoforms in a given spot, depending of course on spot size, density, and thus also quality of resolution [ 96 , 98 , 104 , 105 ]. Therefore, optimal in-gel resolution is needed to ensure the best possible assessment and identification of constituent proteoforms by LC/MS/MS [ 81 , 96 , 102 ].…”

Protein speciation is likely to increase the chance of proteins to be determined in 2‐DE/MS

“…Such shifts in the composition are not attributable to 2-DE/MS per se [3,[6][7][8], which offers the advantage of high resolution down to the content of single spots instead [6][7][8]. The question addressed here is if the occurrence of single proteins in multiple spots might raise their chance of being collected in 2-DE and determined in downstream MS. Leaving aside differential conformation and complex formation of proteins [9], protein speciation due to posttranslational modifications (PTMs) will be a major factor causing multiple spotting of proteins in 2-DE [8,[10][11][12][13][14]. In fact, modifications, such as phosphorylation [15,16], deamidation [17], oxidization [18], and glycosylation [19,20], have the potential to drastically raise the number of protein species which partially will differ in MW and pI.…”

Proteome of Glioblastoma-Derived Exosomes as a Source of Biomarkers

“…Based on this analysis, a list of proteins overexpressed in glioblastoma cells as "potential glioblastoma biomarkers" was generated [13]. We have constructed a database of proteoforms for glioblastoma as well as for normal fibroblasts [14]. Here, we provide a graphical protein pattern (virtual 2DE) of exosomes secreted from five glioblastoma cell lines using high-resolution mass spectrometry (MS) and discuss the presence and the functional roles of the proteins from glioblastoma cells in exosomes.…”