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The term protein species was originally defined by Jungblut et al. [6, 7]. The term describes an individual protein out of the family of proteins coded by one single gene. A protein species is totally described by its amino acid sequence and all posttranslational modifications. In contrast the term "isoform" is clearly genetically defined according to the nomenclature rules of IUBMB [7], describing two or more proteins with the same function but different genes. The large number of proteins arising from the significantly smaller number of genes is caused by RNA processing and protein structure modifying steps (Figure 2). It is estimated that several hundred posttranslational modifications (PTM) exist in eukaryotes [8]. Two basic forms of PTMs can be distinguished. The static PTMs such as oligosaccharides are known to have a key role in protein targeting. Dynamic PTMs like phosphate groups critically determine the activity of a protein. Furthermore alternative splicing can also occur on the protein level. Protein activity is not only determined by PTMs or the action of proteases but also by the interaction of a defined protein species with other biomolecules or ions, binding non-covalently to protein species. For example metallo-proteases require the adequate metal ion to be active. Other enzymes are activated by forming complexes with defined proteins [9]. Further changes to the structure of a protein happen at the end of its lifetime. Ubiquitinylation for example starts its degradation.
Are these differences in the exact chemical structure of a gene product relevant according its function? Since several decades phosphorylation and dephosphorylation of proteins are well known to switch on and off enzymatic activities [10]. Recently it was shown that nitrosylation alters the function of GAPDH radically. Nitrosylated GAPDH is a switch for apoptosis [11]. Truncations of the amino acid chain can activate proteolytic activities of proteases. Binding partners modulate substrate specificity as known from thrombin [12]. Proteins being the product of different splicing on the mRNA level may differ drastically in their functions as was reported from the products of the angiotensin-converting enzyme gene. The protein species synthesized e.g. in endothelial cells is part of the blood pressure regulating system whereas the splicing product present in the testis is involved in male fertility [12].
Figure 2: Protein species: The many protein products of one single gene [7].
Proteomics is defined as the analysis of a defined proteome. The driving force behind the development of proteomics was the hope to gain additional insights into the functioning of a cell or a complete organism by identification and quantification of proteins in different biological states such as disease and health, wild-type and mutant or baseline and perturbed state. The focus on technology development resulted in remarkable improvements in mass spectrometry and coupling MS with liquid chromatography and two-dimensional electrophoresis. The results gained over time in proteomics research have shown that the behaviour and variability of proteins is more complex than ever thought. It is therefore necessary to take the diversification of proteins and the kinetics of their protein species into account to move the field forward. Quantification and resolution of low abundance proteins still remain a demanding task.
The classical strategy, the 2-DE/MS approach, starts with the separation of the proteins followed by enzymatic digestion and identification of the proteins by mass spectrometric analysis of the peptide digest and comparison of the mass spectrometric data with sequence databases (Figure 2, path 2). This approach has the advantage of the separation of proteins at the protein species level and applying the identification at the peptide level, where MS is very fast and accurate. The second strategy (path 1 in Fig.1) starts directly with the digestion of the proteins of a complex mixture yielding a huge amount of peptides, which are separated in the next step, typically via one- or multidimensional chromatographic methods. This procedure is also known as bottom-up approach, because the separation starts already on the level of the peptides. Peptides eluting from a reversed-phase column are then analysed by mass spectrometry (MS) complementing MS with MS/MS-experiments. The latter analysis yields amino acid sequences by which the original proteins can be identified, again by sequence database comparison. The bottom up approach is fast, but does not allow hypothesis-free detection and identification of protein species. To overcome these limitations a mass spectrometric top-down approach (path 3 in Fig. 2) starting with liquid chromatography separations of the protein species and identification of the protein species by mass spectrometry was developed [13]. In the bottom-up/top-down terminology the 2-DE/MS approach represents a top-down separation with a bottom-up identification.
In the first two strategies, the digestion step of the proteins is critical since usually not all peptides of the digest are detected by the mass spectrometric analysis. During the separation steps peptides may be lost by unspecific interactions with surfaces and chromatographic materials. Other peptides arriving at the mass spectrometric detector may also not be identified or cannot be used for the peptide-mass-fingerprinting, if they contain uncommon or complex posttranslational modifications or if they are outside the optimal mass range between 500 and 3000 Da. Furthermore some peptides withstand the desorption and/or ionization process in the mass spectrometer thus yielding no signal in the mass spectrum. As a result the protein identification is in most cases based on a subset of peptides covering significantly less than 100 % of the amino acid sequence of the analysed protein. As a consequence RNA and protein splice variants, proteolytically processed protein species and protein polymorphisms cannot be detected. Furthermore peptides containing posttranslational modifications may be completely ignored. Following the 2-DE strategy this problem is reduced since the proteins are separated in spots and all peptides of one protein are within one mass spectrum, where modifications of the predicted primary structure may be searched. Additionally the sequence coverage can be increased using different digestion procedures. In a bottom-up approach the peptides derived from one protein species are distributed over all fractions of the LC and may be found in any of the mass spectra generated. Even more harmful, peptides with an identical amino acid sequence stemming from different protein species are eluted within one single peak thus making quantification of the individual protein species unfeasible. A hypothesis-free search for modifications is therefore impossible.
Methods & Tools: Protein identification by mass spectrometric methods
Protocols are established for the identification of proteins after tryptic digestion either via peptide mass fingerprint and subsequent MALDI or nano-ESI mass spectrometry. Alternatively tryptic digests are analysed with LC-MS/MS methods applying ion-trap or Q-TOF mass spectrometers. Capillary UPLC and HPLC-chip technologies guarantee low sample consumption. The identification of the proteins from the mass spectrometric data is performed with search engines such as Mascot.
Methods & Tools: Analysis of peptides in body fluids
For the analysis of the composition of peptides in body fluids on- and off-line multi-dimensional liquid-chromatography (LC) systems coupled to mass spectrometry were established (Figure 5). The multi-dimensional online LC-MS system was originally developed by Klaus Unger (University of Mainz) and Egidijus Machtejevas (now Merck, Darmstadt). This systems operates with a chromatographic material termed restricted-access material (RAM) in the first dimension. RAM, developed by Klaus Unger in collaboration with Merck, is applied for concentrating peptides directly from urine or serum by a combination of a size-exclusion and cation-exchange mechanisms. Peptide fractions are eluted towards the reversed-phase separating capillary via a reversed-phase trapping column by a salt gradient.
Figure 3: Multi-dimensional on-line LC-MS system for the analysis of peptidomes. Left: Photo of the system. Right: Scheme of system. 1, 2 & 3: valves. A & B: solvents. MS: mass spectrometer. RAM: Restricted access material: Chromatographic beads with a hydrophilic surface, defined pore-size with a cut-off of 4 kDa and a functionalized surface within the pores. T: Trapping column. W: Waste
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