Insight into Protein Variants/Isoforms and Post- Translational Modifications in a Proteome

Editorial

With the rapid development of human genomics, human structural genome has been completely sequenced accounting for 25,000-30,000 genes [1]. Transcriptomics and proteomics are two important approaches to annotate functions of human genome, or called as functional genomics. However, Transcriptomics and proteomics analyses of the same human tissues reveal that coefficient of relationship of proteome and transcriptome is very low, and the number of proteins is much more than the number of genes [2-4]. It is estimated that the number of human proteins reaches up to over 100,000 or even 1,000,000 if variants or isoforms [5]. It clearly demonstrates that one gene corresponds to multiple proteins are considered which is present as “one gene-multiple proteins” model, but not “one gene-one protein” model [6,7]. Those protein variants or isoforms coded by the same gene are mainly derived from splicing [8- 10] and Post-Translational Modifications (PTMs) [6,11]. Moreover, PTMs are not controlled by genes, and dynamically alter with different conditions such as different physiological conditions, different pathological conditions and different disease stages, etc. Therefore, a gene-coded protein is not only a protein expression event but also involves many post-transcriptional/translational regulations such as splicing, modifications, translocation, and spatial conformation. Those protein post-transcriptional/translational regulations play very important roles in different physiological and pathological processes. Thus, it emphasizes the scientific importance of investigating posttranscriptional/ translational regulations such as splicing, PTMs and spatial conformation in the human proteome.

Since concepts “proteome” and “proteomics” were proposed in 1994 [12], a huge number of publications have focused on protein expression alterations related to different given conditions and proteomic reference maps. Whereas, protein variations/isoforms and PTMs have not been extensively and in-depth studied al all in the field of proteomics, although some PTMs such as phosphorylation [13- 15], glycosylation [16,17], acetylation [18,19], methylation [20,21], nitration [22-24], ubiquitylation [25,26], sumoylation [27,28], succinylation [29,30], sulfation [31,32], myristoylation [33,34], palmitoylation [35,36], deamidation [37], prenylation [38], and hydroxylation [39] have been explored. These mentioned PTMs are only a “window”in the field of PTM database that contains hundreds of PTMs (https://web.expasy.org/findmod/findmod_masses.html; https://www.uniprot.org/docs/ptmlist). Even though phosphorylation and glycosylation are the most extensively studied PTMs; however, only partial phosphorylation- and glycosylation-sites have identified in the human proteome by now. Also, each kind of PTM been dynamically alters with different conditions. Therefore, PTMs that lead to different protein variants/isoforms are very complicated.

Detection and identification of protein variants/isoforms and PTMs in a proteome are essential to clarify biological significance of a given protein variants/isoforms and PTMs in a biological system. For detection techniques, gel and gel-free methods are used [6,7]. Gel-based methods mainly include One-Dimensional Gel Electrophoresis (1DGE), Two-Dimensional Gel Electrophoresis (2DGE), and Two- Dimensional Difference In-Gel Electrophoresis (2D DIGE) [6,11]. Those gel-methods are commonly coupled with corresponding antibody to detect variants/isoforms of a given protein [11], or a kind of PTM [24,40]. For example, 2DGE-based Western blot coupled with growth hormone antibody [11], nitrotyrosine antibody [24,40], and phosphotyrosine antibody [41] is used to detect the corresponding growth hormone variants/isoforms in a human pituitary proteome, tyrosine nitration in an astrocytoma and pituitary proteome, and tyrosine phosphorylation in a glioma proteome, respectively. Gel-free methods mainly include C4 or C5 Reverse Phase Liquid Chromatography (RPLC) with pore size particles of 300 Å [42,43], Hydrophobic Interaction Chromatography (HIC) that is used to separate large bimolecular such as proteins [44, 45], Weak-Cation Exchange Chromatography (WCX) coupled with HIC in a single column with a single phase (2D-LC; from WCX to HIC mode) [46], Capillary Electrophoresis (CE)- ElectroSpray Ionization- Mass Spectrometry (CE-ESI-MS) [47], and multiplexed gel-eluted liquid fraction entrapment electrophoresis (mGELFrEE; size-based separation) with 8 parallel glass gel column [48].

MS is the key technique to identify protein variants/isoforms and PTMs with determination of amino acid sequence of intact proteins, splicing sites [6], and PTM-sites [24,40,41]. Tandem mass spectrometry (MS/MS) can determine amino acid sequence of a protein to directly identify amino acid sequence errors, variations, and modifications, which leads to characterization of protein variants/isoforms and PTMs on different types of mass spectrometers including Fourier Transform Ion Cyclotron Resonance (FTICR) [49,50], Matrix-Assisted Laser Desorption Ionization-Time Of Flight- Time Of Flight (MALDI-TOF-TOF) [51,52], Triple TOF 5600 or 6600 systems [53], and LTQ Orbitrap system [54-56] with different ion fragmentation models such as Collision Induced Dissociation (CID) [57,58], Electron Transfer Dissociation (ETD) [59], and Electron Capture Dissociation (ECD) [60,61]. High-resolution FTICR MS is especially suitable for high-mass accuracy measurement of intact protein ion mass [62]. For analysis of PTMs, a preferential enrichment of PTM-proteins or PTM-enzymatic peptides is necessary prior to MS analysis because of low-abundance of PTM in a proteome and the limited detection sensitivity of MS [22,23,63]. Furthermore, the MS parameter should be adjusted with the features of each variants/ isoforms and PTMs.

Moreover, quantification of protein variants/isoforms and PTMs plays very important roles in understanding their biological significance. Currently, three main categories of quantitative strategies are used: 2DGE-based quantitative methods [64,65], stable isotope-labeled quantitative methods such as isobaric tags for relative and absolute quantification iTRAQ [66,67], and labelfree quantitative methods [68,69] including Selected or Multiple Reaction Monitoring (SRM/MRM) [70, 71] and Sequential Window Acquisition Of All Theoretical Mass Spectra (SWATH) [72,73]. Also, the use of structural proteomics to interpret the spatial conformation of protein variants/isoforms and PTM-proteins would in-depth understand the biological functions of protein variants/isororms and PTMs in a biological system [22,74].

In summary, protein variants/isoforms and PTMs play very important roles in many physiological and pathological processes, and are potential biomarkers and therapeutic targets, which are more promising aspects in the field of proteomics relative to traditional protein expressions. However, studies of protein variants/isoforms and PTMs are much insufficient in the width and depth relative to traditional expression proteomics. Much more efforts should be made to insight into protein variants/isoforms and PTMs in a proteome. Some methods that detect and identify protein variants/ isoforms and PTMs have been developed. However, more effective, accuracy, sensitive, and high-throughout detection and identification techniques are needed to maximize the coverage of protein variants/ isoforms and PTMs in a proteome.

Acknowledgement

This work was supported by the grants from China “863” Plan Project (Grant No. 2014AA020610-1 to X. Z.), Xiangya Hospital Funds for Talent Introduction (to X.Z.), the National Natural Science Foundation of China (Grant No. 81272798 and 81572278 to X.Z.), and Hunan Provincial Natural Science Foundation of China (Grant No. 14JJ7008 to X. Z.).

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