Free Research Paper About A Review Of Mass Spectrometry Techniques Used In Diabetes Screening.
Diabetes is a disease caused by high blood glucose levels due to underproduction of insulin in the body or cell resistance to already produced insulin. The disease is characterized by hyperglycemia that leads to non-enzymatic protein glycation. Mass spectrometry techniques have been developed to give highly specific, reproducible and reliable results in the proteomics field especially in monitoring the progression of diabetes, the complications that arise from it, and the effectiveness of therapeutic treatments. An overview of mass spectrometry and current methodology such as matrix-assisted laser desorption ionization (MALDI) indicates that the technique has certain limitations in terms of quantification applications, reproducibility and analysis of low mass analytes. The papers also discusses some state of the art methods such as Liquid Chromatography/Tandem Mass Spectrometry, and collision and fragmentation techniques along with an example application of LC-MS/MS to determine 25-Hydroxy Vitamin D2 and D3 percentage in Dried Blood Spots for T1DM screening. Novel techniques devised to help counter the quantification and reproducibility limitations of MALDI are also discussed along with future perspectives on identifying new biomarkers for MS analysis and the importance of analyzing new diabetes biomarkers to get more accurate information in the early stages of diabetes pathogenesis.
Diabetes refers to a group of metabolic diseases characterized by high blood glucose levels (hyperglycemia) resulting from underproduction of insulin from the body or lack of cell response to the insulin produced. The hyperglycemic conditions induced by diabetes lead to non-enzymatic protein glycation responsible for chronic complications. There are three main types of diabetes i.e. type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), and gestational diabetes mellitus. Type 1 diabetes arises from the body’s failure to produce enough insulin while type 2 arises due to the body’s resistance to the produced insulin. Although diabetes is still regarded as an incurable condition with significant implications for human health, the availability of genetically engineered human insulin has made it possible to control diabetes. However, various research efforts conducted over the years suggest that the hyperglycemic conditions of diabetic patients might be greatly heightened of even reversed if the disease was identified and controlled in its early stages. In this case, early diabetes detection and diagnosis has become a topic of significant interest to researchers aiming at preventing and treating the disease .
The development of mass spectrometry techniques has made it possible to give highly specific, accurate and reliable results in proteomics. These techniques have therefore become of great interest to researchers and physicians by providing them with new tools to diagnose and monitor disease progression, discover possible diabetes-related complications, and identify the effectiveness of the applied therapeutic treatments. This paper provides a review of mass spectrometry techniques, their application in proteomics, limitations, state-of-the-art methods, and novel experimental approaches all with specific interest in diabetes pathogenesis.
Overview of Mass Spectrometry and Proteomics.
Mass Spectrometry (MS) is an analysis technique used to measure the ratio of mass to charges in order to identify and quantify molecules in both simple and complex mixtures. MS has widespread applications in various fields but for the scope of this paper, only the proteomics field is considered.
Proteomics is the study of proteins i.e. cells, tissues and organisms in a biological system during certain biological events. Proteomics complements genomics but is significantly more complex due to the dynamic nature of protein expression. Additionally, most proteins go through some form of PTM (post-translational modification) that further increases the complexity of proteomics. Over, the last two decades or so, high-throughput and qualitative workflows in MS proteomics have been developed thus expanding the scope of what is known about the structure of proteins, functions, modifications and overall protein dynamics .
Mass Spectrometry is a high throughput technique used in the detection, identification and quantitation of molecules on the basis of their mass and charge (m/z). The technique was originally developed over a century ago to measure the atomic weights of elements and the natural abundance of particular isotopes. In biological sciences, the technique was first used for tracing heavy isotopes through bio-systems and later for nucleotide structure analysis and sequencing of peptides and oligonucleotides .
Development of macromolecule ionization methods such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have enabled MS protein structure analysis which has helped scientists establish unique protein mass identifiers that can be matched to peptides and proteins in databases to help determine the identity of unknown proteins. Isotopic tagging methods have also enabled both relative and absolute target protein quantitation. Further technological advances have led to the development of sample analysis methods for gas, liquid, and solid states. Current mass spectrometers are so sensitive that they detect target molecules at attomolar (10-15 mol/m3) range concentrations .
In proteomics, mass spectrometry is used to identify proteins from the mass of peptide fragments, quantitate proteins in a given sample, and to determine protein structure, folding, functions and interactions. Other applications include detection of specific post-translational modifications in complex biostructures and to monitor chemical modifications, enzyme reactions and protein digestion .
Components of a mass spectrometer.
Mass spectrometers have three basic components, an ion source, the mass analyzer and ion detector. However, the nature of these components varies with the type of spectrometers, technology used, type of data required and the samples’ physical properties. Samples can be loaded onto the spectrometer in dry or liquid form then vaporized and ionized by an ion source e.g. MALDI and ESI. The Figure 1 below shows the basic components of a mass spectrometer.Figure 1 the basic components of a mass spectrometer.
The charge received by the molecules allows the ions to be accelerated through the rest of the system. The accelerated ions encounter magnetic and/or electrical fields from the mass analyzers which deflect them onto individual paths based on their mass to charge ratio (m/z). Commonly mass analyzers include ion traps, quadrupoles, and time-of-flight (TOF). Each type has its specific characteristics and application. Mass analyzers are also used for separating all analytes in a sample for use in the global analysis, or they can also be used to filter and deflect a specific group of ions to the detector. Once the ions have been deflected successfully by the mass analyzers, they hit the ion detector. Most detectors comprise of micro-channel plates or electron multipliers that emit an avalanche of electrons as each ion hits the plate. The avalanche then results in amplification of each hit ion to improve sensitivity. The entire process is conducted under extreme vacuum conditions (10-6 to 10-8 torr) to eliminate non-sample contaminant ions that could collide with sample ions thus altering their path or producing unwanted reaction products. Mass spectrometers are linked to computer systems that analyze the detected ion data and produce charts that organize the detected ions based on their individual mass-to-charge (m/z) ratio and their relative abundance in the sample. The results of this analysis are run through databases that predict the identities of molecules based on their m/z .
2. Problems and Limitations with current Mass Spectrometry methodology.
Over the years, mass spectrometry (MS) has been used in the investigation and analysis of non-enzymatic protein glycation for diabetes screening and pathogenesis. The high specificity and sensitivity of MS techniques facilitated the development of methods that can characterize and analyze glycation biomarkers to be validly applied in diabetes monitoring. A good example of well-developed and currently used technique is matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) . Since its inception in 1987, the technique has been used to analyze a wide variety of biomolecules and to determine the molecular weight of intact proteins. It was first used in the study of in vitro reactions between different proteins and hexoses and once the results of these tests were affirmed to be valid, a furore of investigations on plasma proteins obtained from both diabetic and healthy patients was launched. The method led to the study and evaluation of the number of glucose molecules condensed on the plasma protein. Since then, the method has been validly applied in the accurate following up of metabolic control in diabetes patients. When MALDI was applied to hemoglobin glycation studies, the results indicated that the extent of glycation on both alpha- and beta- globin was the same, and that simply glycated molecules have glycol-oxidized species accompanying them thus providing information on the subject’s oxidative stress that was being experimented on.
When immunoglobins were analyzed, MALDI/MS determined the total glycation levels of IgG and also helped establish that the most glycated one was the fragment antigen binding (Fab) moiety. These findings suggested that the immunological impairment sometimes invoked in diabetes is related to inhibition of the molecular recognition process between antigens and antibodies . Other initial applications were almost exclusive on qualitative analysis of biopolymers because MALDI-TOF MS provided a quick and highly accurate approach to acquiring molecular mass and purity data i.e. checking for material for the right mass and presence of contaminants. Due to its high analysis speeds, ease of use, relatively low cost of equipment, limited potential for sample cross-contamination, and ease of data interpretation, MALDI-TOF MS systems were considered as walk-up tools where researchers could run quick samples to determine if their work was on the right track .
However, despite being a much acclaimed MS method, MALDI-TOF MS was considered impractical in two specific application areas i.e. quantitative applications and analysis of low mass electrolytes. Quantitative analysis was considered far-fetched since crystallization does not yield uniform distribution of analytes and matrix on the target surface thus giving rise to certain regions on the target surface where analyte concentration/signal was higher compared to other regions on the same surface. Low mass analysis, on the other hand, has complexities which are attributed to the vast molar excess of the matrix which can deluge any analyte-specific signal in the low mass-to-charge (m/z) region of the spectrum. With the above two considerations, results from MALDI MS analysis were considered irreproducible since each given amount of analyte loaded onto the target would register a different value when ion intensity was measured . However, new methods and reports of experimentation indicate that the perceived limitations of MALDI can be overcome and that quantification can be made routine for both high and low mass analytes. These methods are discussed later in section 4.
3. State of the Art MS Methods: Tandem mass spectrometry (MS/MS):
The Tandem mass spectrometry (MS/MS) approach is now widely used in proteomics especially in diabetes screening since the technique provides extra information about particular ions. In this approach, the specific ions of interest are selected on the basis of the m/z ratios from the first MS cycle and then they are fragmented using one of the several dissociation methods such as ion collision with inert gas streams also known as collision-induced dissociation (CID), and higher energy collision dissociation (HCD). Other methods used to fragment ions include electron capture dissociation (ECD) and electron transfer dissociation (ETD). The now dissociated fragments are separated in another MS round based their individual m/z ratios. The MS/MS technique is widely used in oligonucleotides and protein sequencing since the fragments can be used to match predicted nucleic acid or peptide sequences, respectively, which can be found in databases such as Swis-Prot, RefSeq, and IPI. The sequence fragments can be organized in silico to predict full-length sequences .
Methods of analyte separation: Chromatography.
Biological samples can be quite complex often containing molecules that can mask the detection of the target molecules. This usually occurs when a wide dynamic concentration range is exhibited between the target analytes in the sample and the other molecules. Various separation methods are usually employed to split up the target analytes from other molecules in the sample. Some of the methods include gas chromatography (GC) and liquid chromatography (LC) which are used in the analysis of complex gas and liquid samples by mass spectrometry, respectively .
HPLC (High performance liquid chromatography) is the most common state of the art method of separation used in the analysis of biological samples by MS or MS/MS (or LC-MS and LC-MS/MS respectively) since most biological samples are liquid and non-volatile in nature. The small diameters of LC columns (e.g. 70μm, nanoHPLC) coupled with their low rates of flow (e.g., 220nL/min), make them ideal for analyzing minute samples. Additionally, LC can be linked directly to MS (in-line LC) to provide a high-throughput sample analysis method. This approach works in such a way that multiple analytes eluting through the LC column at different rates are analyzed immediately using MS .
Example of state-of-the-art LC-MS/MS method used to determine 25-Hydroxy Vitamin D2 and D3 in Dried Blood Spots for T1DM screening.
Apart from determination of protein and peptide glycation using MS for diabetes screening, screening for Vitamin D deficiency has emerged as a new technique for identifying risk of type 1 diabetes quite early in its pathogenesis. The technique in question is the LC-MS/MS method for 25-Hydroxy Vitamin D2 and D3 in Dried Blood Spots developed by Newman et al.  that is currently under evaluation with high possibility for future clinical applications.
According to Newman and colleagues , the of vitamin D deficiency is a possible cardio-metabolic risk factor for type 1 diabetes especially in consideration that it is also associated with metabolic syndrome, obesity and type 2 diabetes. Newman et al.  deduced that an accurate screening test for deficiency in 25-Hydroxy Vitamin D [25(OH) D] was all that was needed and they developed a LC-MS/MS assay for 25-hydroxy vitamin D2 and D3 i.e. [25(OH) D2] and [25(OH) D3] in dry blood spots .
Blood spots and serum samples were simultaneously obtained from healthy volunteers using a finger stick and venipuncture. Disks were then punched from the dried blood spots and then sonicated using a standard solution of deuterated 25(OH) D3 (i.e. replacement of hydrogen with heavier element deuterium). Methanol was then added to facilitate protein precipitation before they were extracted using hexane. The sample extracted were dried and reconstituted in a 50:50 mixture of H2O and Methanol before being injected into a Varian 320-MS TQ mass spectrometer.
The precision of the blood spot assay was good and well over the reportable range. The inter-assay coefficients of variation reported were 13%, 13% and 11% at concentrations of 14, 26 and 81 ng/ml respectively for Vitamin D3 (25(OH) D3), and 12% for Vitamin D2 (25(OH) D2) at a concentration of 23ng/ml. The Vitamin D3 assay was linear at concentrations of 3.5 to 75 ng/ml (R>0.99). The blood spot and serum values obtained also demonstrated great correlation for 25(OH) D3 (R = 0.91, n = 83), and 25(OH) D2 at (R = 0.90, n = 54)  as shown in Figure 2.
Figure 2: the correlation between blood spot and serum levels of 25(OH) D2 and 25(OH) D3 from simultaneously collected samples from healthy volunteers. The solid line is the Deming fit while the dotted line is the identity (Source: Newman et al. ).
4. Novel experimental approaches to deal with current MS limitations:
As seen in the previous sections, Tandem MS (MS/MS) in combination with fragmentation techniques, and LC or GC have high specificity and accuracy since more details can be deduced about an ion than other MS techniques such as MALDI. However, despite the apparent limitations of MALDI in terms of quantitative applications and analysis of low mass analytes, there have been several approaches made to improve on MALDI.
Researchers have gone as far as creating variants of the basic MALDI approach with modifications centered on the matrix and its application. Some modifications also offer potential quantification benefits. For example, desorption/ionization of silicon (DIOS) allows for protein and small molecule analysis in the absence of a matrix. Just like MALDI, DIOS affords little or zero fragmentation and has relative tolerance to moderate amounts of contaminants often found in biological samples. The lack of a matrix presents special benefits especially in the low mass regions .
It has also been reported that use of ionic matrices in the quantitative application of small proteins, peptides and oligodeoxynucleotides produced better calibrations and achieved better linearity and reproducibility over a wide range of concentration for the tested ILMs (ionic liquid matrices) despite the different physical states. However, it is was also noted that the standard deviation was higher for ILMs compared to solids with visible crystals .
Another MALDI modification is reviewed in  where it is reported that the use of an acoustic reagent multi-spotter helped provide improved reproducibility for the deposit of matrix onto a sample surface. This technique was originally developed for tissue section imaging, but it may also be used in quantification techniques used in diabetes screening. The acoustic droplet ejector was observed to provide better control of conditions that affect matrix crystallization and protein extraction thus offering the ability to deposit matrix accurately onto small surface features. The quality and reproducibility of MS using the acoustic multi-spotter was found to be better than when spotting is using manual pipettes.
Other methods used to improve on MALDI-MS include the use of dry prepared sample methods otherwise known as solvent-free methods that have shown the superiority when compared to traditional wet sample preparation techniques. For example, dry sample preparation techniques allow for the analysis of insoluble samples and reportedly deliver better quality mass spectra. Overall, there have been other numerous modifications on MALDI-MS to improve on quantification precision and accuracy, but are still to be rigorously assessed in a quantitative setting to ensure their viability.
5. Future Perspective in Mass Spectrometry and diabetes:
Current diagnosis of diabetes is based on persistent or recurrent hyperglycemia and in a bid to find new diabetes biomarkers, research shows that neither glycated hemoglobin (hbA1c) nor plasma glucose levels can be used for early diabetes detection. Plasma glucose levels are easily affected by the lifestyle adopted by the patient i.e. body condition and food intake. HbA1c levels, on the other hand, have a poor quantitative relationship with glycation accumulation and low sensitivity .
In this regard, new approaches are being made to identify new biomarkers which are currently being explored, and these include alanine aminotransferase, plasminogen activator inhibitor, triglycerides and C-reactive protein. Enzyme-linked immunosorbent assay (ELISA) has been used widely to detect diabetes biomarkers. ELISA is highly sensitive due to the high catalytic efficiency attributed to enzymes, but it also requires prior knowledge of biomarkers and the corresponding antibodies. Advanced glycation end products (AGEs) serum concentration is also a known marker for monitoring diabetes complications treatment and can be detected using fluorescence. Aside from the protein level biomarkers, peptide level biomarkers such as C-peptide have also increased in popularity .
Mass Spectrometry based quantitative proteomic methods i.e. relative and absolute quantitative proteomics are applied in the biomarker exploration and diabetes pathogenesis. The absolute quantitative method uses a standard curve for quantification of target proteins. One of the novel diabetes biomarkers, C-reactive protein, has been quantified using MS both in the presence and absence of an affinity removal system .
Relative quantitative proteomics has been used in the exploration and mechanism studies of diabetes biomarkers. Using isotope labeling methods, Zhang et al.  showed that S-nitrosation could be involved in T2DM pathophysiology. Glycation isotopic labeling methods have also been used to monitor different hemoglobin glycation states in vitro. Zhang et al. [4-6] also designed a bottom-up proteomics approach applying electron transfer dissociation tandem MS (MS/MS) and boronate affinity chromatography analysis in the study of peptides and glycated proteins. Zhang and colleagues also reportedly identified 7,749 unique glycated peptides using a similar process after eliminating 12 highly abundant plasma proteins .
Non-enzymatic protein glycation has recently attracted more attention towards research in proteomics due to its clinical applicability to diabetes and related complications. Glycation does not have chemical selectivity as a non-enzymatic reaction which means that highly abundant proteins are more susceptible to modifications by plasma glucose. Consequently, quantifying glycation levels of highly abundant proteins using MS analysis is much more sensitive than testing plasma glucose while screening for diabetes .
Zhang, Xu and Deng , have developed a standard free and label-free MS-based proteomics method to be used in early diagnosis of T2DM. Human serum albumin (HSA), the most abundant plasma protein (~62%) was used as a glycation collector and monitored by quantitatively analyzing the characteristic peptides in HSA. According to Zhang, Xu and Deng , this strategy differs from the traditional concept that almost all proteins are low abundance and that disease biomarkers should be screened for low abundance proteins. In fact, the use of high abundance proteins as the target analytes increases the accuracy of quantification because the glycation of highly abundant proteins is dominant in vivo, and the use of a highly abundant target improves sensitivity of MS by reducing detection errors. Another reason why research on peptide biomarkers should be considered for the future of diabetes screening instead of protein biomarkers that are currently used is because proteins in vivo undergo various types of post-translational modifications such as phosphorylation, methylation, glycosylation, acetylation, and ubiquitination. Each one of these modifications can significantly alter the molecular weight of target proteins leading to difficulties in MS analysis .
While the number of known diabetes biomarkers is quite large, each has its limitations in terms of sensitivity, capability, and overall feasibility, and are, therefore, subject to further research and possibly clinical applications in the future.
The information presented in this paper shows that mass spectrometry can be considered as valid analytical tool for diabetes screening especially in the study of non-enzymatic glycation of proteins. Other indicators and biomarkers such diabetes screening through determination of Vitamin D deficiency and the emphasis on discovering diabetes biomarkers have also been discussed along with future research prospects for use in diabetes screening via MS. It has been seen that the current methodology such as MALDI has its limitations, but some modification techniques to improve on this method have shown key promise and produced key result. In this regard, it should be noted that MS techniques are undergoing continuous improvements and more innovative and accurate methods are being devised through collaboration between scientists, physicians and instrument manufacturers alike to ensure that diseases such as diabetes are monitored and discovered early enough to avoid future complications.
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