Overview on capillary electrophoresis with mass spectrometry: Application in peptide analysis and proteomics

Introduction

An increasing demand for sensible and selective protein analysis analytical techniques are due to recent development in protein chemistry, proteomics and biotechnology. Capillary electrophoresis (CE), slab gel electrophoresis (SGE) and Liquid chromatography (LC) are most popular separation techniques used for qualitative and quantitative determination of protein analysis.^[1] As several samples can be studied simultaneously, SGE is a well-established technology for protein separation and a relatively large number of proteins can be solved using 2D SGE. However, restrictions include relatively long, labour-intensive analysis, the need for offline detection and the absence of precision (and automation). The benefits associated with SGE are easy automation, separation capability, and routine linkage, including mass spectrometry (MS) is advantageous for different detection concepts.^[2] On the other hand, because of the complex nature of the proteomes, adverse interactions between proteins and the stationary phase and protein denaturation can seriously impede LC studies in the organic mobile phase. This can decrease the separation efficiency and resolution. Even when proteins and peptides are large biomolecules, column blocking can be a concern.^[3]

Short analysis time and limited sample requirements for protein analysis are the desirable characteristics associated with CE. Furthermore, in the case of fused-silica capillaries and without a stationary phase, CE tests are carried out under aqueous conditions. This enables protein to be analysed by organic modifiers and/or a stationary process without creating changes to conformity. In addition, CE divider conditions may be selected to avoid protein degradation during analysis under (near) physiological conditions. In conclusion, as CE separation is a characteristic of the compound's loading, size and shape, with small differences in protein size or load necessary to separate the compound, especially with a high CE efficiency. MS has become one of the most popular and useful techniques for small fragment detection.^[4], ^[5]

CE-MS has therefore become an efficient analytical technique that allows the separation speed to be combined with the selectivity and the structural information given by the MS, high-resolution capacity and minimal sample consumption of CE.^[6] Several key advances in CE-MS technologies over the last 10 years have made CE-MS a competitive tool to solve various analytical problems.^[7], ^[8], ^[9], ^[10], ^[11], ^[12], ^[13] The application of CE-MS is not limited to peptide analysis, now a day’s modern CE-MS method are also used for the profiling of metabolomics.^[14]

In various fields of information, study of chiral molecules is of particular importance. The pharmacological or toxicological properties of enantiomers may be different, making CE-MS one of the methods of choice for this type of assay. Furthermore, Armstrong et al. have suggested that enantioselective separations can be used in food and beverage studies for the identification of adulterated foods and beverages, monitoring of fermentation processes and products, age assessment and identification, treatment and storage effects, and evaluation of some co-products of flavor and fragrance. ^[15]

In the pharmaceutical and biopharmaceutical industries, glycosylation studies (adding a carbohydrate unit to a protein molecule) play a pivotal position. Therefore, study and systematic profiling of such glycoproteins is a crucial parameter for determining their biological activity and protein stability. In this analysis, which is the usual post-translational modification of proteins, the CE / MS approach helps. A effective instrument for the impurity profiling of drugs such as tetracosactide is the CE / MS technique. CE / MS in pharmaceutical review is a modern-day saviour. CE is essential for quality management tools for pharmaceutical entities and products. In the study of intact proteins, CE / MS also has applications, either in the development of methods or in the characterization of biopharmaceutical formulations. In the analysis of analytes such as Recombinant Human Insulin, Growth Hormone, Haemoglobin etc., CE / MS helps Characterization of Metal Protein complexes in another modern CE / MS programme that lets the analyst evaluate the metal protein complex's stoichiometry.^[16]

In the study of intact serum proteins, the CE / MS study also has applications that are especially useful for the study and characterization of plasma drugs and their binding to plasma proteins. In biomarker discovery and application, capillary electrophoresis coupled with MS has been successfully used, as it allows the selective detection of small protein peptides. CE-MS enables extremely complex samples to be characterized (such as urine, plasma and other bio fluids). It has a number of uses, many of which concentrate on studies of urinary peptide biomarkers in renal and cardiovascular diseases in particular. The malignancy of the genitourinary system has been another important area of concern. Therefore, CE / MS has tremendous potential for cancer patients in the area of biomarker discovery and clinical application.^[17]

In a single amino acid, the majority of variant proteins differ from normal proteins. Peptide maps or "fingerprints" have commonly been used to classify the locations of alteration. Both the direct method and the peptide mapping method of LC- MS have recently been developed as powerful tools for analyzing protein structures and mutations.^[18] It is very appealing to combine the high performance and high speed of CE with the high sensitivity and high selectivity provided by MS detection. CE is very tolerant of complex sample matrices and thus allows for the highly selective identification of compounds in several complex mixtures when combined with MS. In suitable cases, MS identification also helps to enhance the general sensitivity of CE analyses. The strength of incorporating MS detection with any hyphenated approach is that it provides a second dimension of separation. MS is then divided first on the basis of the charge-to-size ratio of the analyte and then, in accordance with capillary zone electrophoresis (CZE), on the basis of its m/e ratio. A number of methods of ionisation and coupling have been attempted (it is important to improve methods of ionisation and coupling).^[19] The most popular method for routine coupling of CE with MS is electro spray ionization (ESI).^[20], ^[21], ^[22]

High separation speed of CE coupled with MS detector becomes efficient techniques widely used in food analysis. ^[19] In general, the analysis of analytical results can be clearer if a separation strategy is combined with MS.^[11], ^[13] As a result, the technique of electron spin ionisation has emerged as a highly useful technique that allows direct linkage with electrophoretic separation techniques. ^[23] To obtain fragments of highly complex target compounds, collisionally induced dissociation can be used. The Instrumentation of CE with MS is shown in [Figure 1].

MS identification for CE has been aggressively sought in recent years, because MS is deemed to be more universal than UV absorption. MS's selectivity and accuracy compensate for differences in the analytes' migration times, an issue frequently encountered in CE. Apart from molecular weight and structural information, MS detection also provide information about co-eluting fragments of the target compounds which leads another dimension of separation efficiency of the CE-MS. Low attomole to femtomole concentrations can be detected by MS hyphenated with other efficient separation techniques, which simplify the analytical result with increasing detection limit. Due to these advantages, CE-MS is widely reported technique for peptide analysis compared to other available techniques.^[24], ^[25], ^[26]

The CE/MS coupling has achieved a substantial maturity stage in the past few years. CE / MS interfaces and applications are detailed in several review papers. This report highlights the significant advances in CE / MS that have taken place during the last four years.^[9], ^[27] Applications using some of the more common electrophoretic separation modes have been illustrated, such as classical electrophoresis of the capillary zone (CZE) and capillary isoelectric focusing (cIEF). Latest work on capillary electro chromatography (CEC)/MS is also being considered.^[28], ^[29] A variety of different types of mass analyzers have been coupled to CE, including triple quadruple (QQQ), Fourier transform ion cyclotron resonance (FT-ICR), ion trap (IT) and time of flight (TOF).^[30], ^[31] ESI is considered to be the method of choice for interfacing CE with MS because it allows molecules to be transferred directly from the liquid phase to the gas phase.^[32], ^[33] As a unique characteristics given by CE include low consumption of samples/reagents and flexibility, fast analysis. Therefore, CE-MS has become an effective separation and identification technique for the complex peptide and other biomolecules analysis.^[34], ^[35], ^[36], ^[37], ^[38]

Applications of CE-MS in Peptide Analysis

Capillary Zone Electrophoresis - Mass Spectrometry

Smith et al. demonstrated that proteins could be readily analysed with CZE-ESI-MS. Their research was the point of departure for the discovery and application of CZE with MS detection for intact protein analysis. Below is an overview of the CZE-MS protein that allows for subdivision into interfaces based on ESI, MALDI and ICP.^[69]

Electro-Spray Ionization

The CZE is the simplest CE mode while in protein MS, ESI is most popular. Not surprisingly, in intact proteins, CZE-ESI-MS is the most widely used in CE-MS. CZE-ESI-MS was first applied to analyse model proteins. Later, its uses grew to include more complex analytical problems like isoform identification, biomarker screening as well as single cell study. To recognise potential biomarkers for some diseases, Mischak et al. worked and published various studies on the screening of different body fluids. Urine ^[70] was primarily investigated, but serum, ^[71] plasma ^[72], ^[73] and cerebrospinal fluid were also analysed. The CZE-ESI-MS data were provided by plotting migration times against their molecular masses of the observed constituents, resulting in two-dimensional graphs. Possible biomarkers were identified by comparing the graphs of diseased and healthy volunteers.

Because of the comparatively low concentrations, compared to hemoglobin, the study of carbonic anhydrase I and II in RBCs is very difficult. The CZE-ESI-MS haemoglobin and carbonic anhydrase studies have been demonstrated by Moini et al. ^[74] The haemoglobin and carbonic anhydrase I chains were possible to separate and detect, but carbonic anhydrase II chain was not detected due to low concentration.

Stutz et al. have developed CZE-ESI-MS method in which different clinically important metal binding protein pre concentrated by iso-tachophoresis protein steps. Where they could succeed to identified five metal binding proteins by using background electrolytes such as formic acid with detection limit lower to sub-femtomole per litre. This method seems interesting, but has not been used for actual samples until now.^[75]

CE-MS Using Bare Fused Silica Capillaries

A good technique for peptidomics has been shown to be CE-MS. So, the discovery of biomarkers is play vital role in peptidomics. As peptides have already been shown, certain ongoing malfunctions or diseases of the organism could be revealed.^[37] Therefore the identification of new biomarkers is very important to aid in diagnosis.^[70], ^[76] A CE ESI-TOF instrument was used by Kaiser el to determine a polypeptide pattern. The study of patterns may not only suggest differences in certain diseases, but also in the stage of a disease. CE-MS has been shown to be an important analytical tool that could substantially lead to an unambiguous diagnosis based on polypepide pattern analysis, the authors say.^[77] Protein identification through analyses of peptides produced following the biopolymer's enzyme digestion including analysis of protein modifications is one of the main uses of CE-MS in proteomic/peptidomics research. ^[78], ^[79]

The normal generation of unintended peptide fragments presents a major limitation to overcome this issue, the use of theoretical models to simulate peptide activity in CE may be very useful. It has been demonstrated that the use of CE-MS in combination with bare silica capillaries is a very useful analytical tool for the study of peptide hormones. It can also be used to identify microorganisms in small quantities.^[80]

A theoretical model was also developed to connect the sequence of peptides effectively with their electrophoretic behaviour. The efficacy of this model was demonstrated by the huge success in CZE-MS in analysing all of the peptides obtained in one cycle. This model was later examined by comparing the particulars of its slitting to investigate the equivalence of natural and recombinant enzymes.^[81], ^[82], ^[83]

Chip-based electrophoresis combined with mass spectrometry

New CE-MS technology based on chips is also a growing field, as miniaturisation is now a major focus for scientists. The CE on a chip can generally be highly flexible in combining various analytical steps, shorter analytical times, lower sample and reactant consumption and high efficiency. At the same time as this great interest in chip-systems new devices are being developed to connect them to MS instruments.^[83]

Karger and colleagues have therefore used on-chip CE-MS method demonstrating a modern concept including sample inlet ports, separation channel, interface chip and liquid junction MS and electrospray capillary insertion guide channel. Where peptides were chosen to evaluate efficiency of the developed system and peptides were readily separated in a 4.5 cm on-chip separation tunnel in the region of attomole with achievable detection limit.^[82], ^[83] Combination of micro fluidic CE systems with mass spectrometric detection is one of the best chip based analytical tool, where sample analysis speed and efficiency can be increased with low cost, weight and size of instrument.^[84], ^[85] The main application of chip-based CE-MS method is protein digestion in the field of peptide analysis with intact protein analysis has also been mentioned in some cases. CZE /CIEF and ESI-MS / MALDI-MS integrated on-chip CE combined with MS are generally adopted method for intact protein analysis. Whereas glass,^[81], ^[86] quartz^[87] polycarbonate,^[88] polymethyl methacrylate and polyester are used as chip material, mostly in combination with capillary coatings to minimize protein adsorption.

CE-MS using on-line pre-concentration techniques

A crucial problem associated with CE-MS peptidomics is the adequate sensitivity to carry out study with low concentrations of these peptides in real samples. In the CE-MS coupling, due to several factors, the good sensitivity given by MS instruments is reduced.^[89]

  The capillary can inject very low samples, without affecting the efficacy and resolution of the separation and, as a result, the ESI interface is reached by a very limited number of analytes.

  The constant need for additional sheath liquids to obtain a safe spray which reduces the sensitivity due to frequent dilution

  As a loss is commonly observed, the effect of ionisation and solution transfer to the MS analyzer must also be taken into account.

To address above limitations, different techniques have been developed to pre-concentrate the sample before ESI-MS analysis, whereas sample stacking is one of the best technique.^[89] For peptide analysis, some on-line pre-concentration devices based on solid phase extraction (SPE) were also used.^[90] Thus, Janini et al. created new design with capillary of 20 μm ID using one of these devices for a regular peptide mixture as shown in [Figure 2]. Where peptide analysis can be done within 15 min and SPE cartridge can easily replaced.^[91]

Chien and Helmer have proposed first field amplified sample injection system as another pre-concentration process used in peptidomics. It is based on the use of two separate buffer solutions for filling the capillary separation, typically at the identical pH with varied concentrations. Under these conditions, more than 3000 times the sensitivity increase was obtained as a result of this injection procedure. Sub-nanomolar concentration ranges for the peptides studied could be detected.^[92], ^[93], ^[94] Larsson and Lutz were able to raise the injection volume by up to 0.9 μl, where they used transient iso-tachophoresis (transient-ITP) technique to address the low sensitivity issue in CE-MS. It is possible to increase the injection volume substantially by using this technique without losing performance and resolution.^[94]

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