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Describe the sample collection

1. The metabolomic information we get and the conclusions we can draw are largely dependent on the biological samples collected. It is thus of prime importance that collecting and extracting the samples is handled very carefully/ Tissues, biofluids and primary cells are the three different kinds of samples that can be collected (Metabolomics, 2019). Saliva, Plasma, Serum, Cerebrospinal fluid (CSF) and urine are the different types of biofluids. These can further be divided into metabolically active and inactive samples. Biofluids like urine fall in the category of inactive samples as they are extracellular while samples of plasma/serum, CSF and saliva are considered metabolically active (Metabolomics, 2019).

There is a quantitative and qualitative difference in results, which emanates from the time at which metabolically inactive samples, specially urine is collected. The best option is to collect it during the early morning void and then use liquid nitrogen to freeze the sample at -80 degree centigrade so that it doesn’t degrade (Silva, Passos & Câmara, 2012). This would also prevent the contaminated bacteria from metabolising the sample.To enhance the efficacy, prior to chemical derivatization the sample has to be lyophilized using chemical compounds like BSTFA and MSTFA which are used to derivatization of urine. To stabilize the sample that needs to be stored MSTFA is used (Blondal et al., 2013). Nowadays, there are instances where solid phase extraction and liquid-liquid extraction are used as step to ensure clean up. This also helps exclude derivatization. Headspace solid phase microextraction (HS-SPME) utilization has been implemented effectively in testing unstable organic compounds in urine (Metabolomics, 2019). The heating of the urine will result in the volatile compounds entering the gas phase. They will then get attached onto the solid phase microextraction fibre before they are delivered to the GC-MS instrument (Blondal et al., 2013). The major advantage of using the SPME procedure is that it comes without the requirement of any dissolvable and is a faster way. It also helps add a sample concentration step (Blondal et al., 2013). It is cheap and doesn’t have any negative biological impact. To prevent urea in the urine to severely affect the data quality in GC-MS test, catalysing by enzyme urease is done. This helps in detecting low abundance metabolites. Using urease enzyme was found out to be destructive in a few instances too (Silva et al., 2012).

To sum it up, it has been found in several studies that to diminish the effect of fluctuation of the concentration of urine on biological results, the concentration of urine can be equalized when the sample is being prepared. To equalize the sample, osmolality or specific gravity are techniques that can be used to measure the concentration (Silva et al., 2012).

2. In the metabolomics stage, Gas chromatography (GC), which is a traditional procedure is used in combination with certain types of mass spectrometers. This technique is based on the separation of unstable and those metabolites which are thermally stable (Sugimoto et al., 2012). Esters, ketones, aldehydes, alcohols are some naturally stable compounds while volatile compounds consist of those compounds made by derivatizationare sugar phosphates, lipids, sugar, amides, amino acids and amines (Allied Academies Conference, 2019).

Once the sample is taken in the GC instrument, metabolites are volatilized instantly. These samples are then transported to a coated capillary column by inactive gases nitrogen or helium from a heated infused system (200-250 degree centigrade) (Jasim et al., 2015). There is a phase called stationary phase which is a column made by solid or liquid phase. The metabolites which are in an inactive gaseous state are made to pass through this phase. The temperature of the coated column is maintained by the incinerator with the aid of a temperature regulator (Altameme et al., 2015). When the temperature increases, it is seen that composites with low boiling point saw extraction before the composites with high boiling point. The separation of the metabolites between the temperature and the solid or liquid phase is caused due the chemical interaction (Altameme et al., 2015). When a mass spectrometer is linked to the dissociation of the metabolites, it shows increased sensitivity and power. Gas chromatography has its own limitations. Some metabolites are not volatile naturally and hence is requires derivatization which consumes time and is prone to mistakes (Jasim et al., 2015). The process of derivatization is done in two phases that involve oximation and silylation/chloroformate reagent. Oximation has distinct advantages as it stops the functional groups of ketones from tautomerism as well as carboxylation and also prevents the development of the rings of sugars (Altameme et al., 2015). It is pertinent that the sample completely dries out to prevent hydrolysis of the reagents preceding sylation. Using Chloroformate as a reagent assures that there is no conduction in the aqueous media. The mass spectrometer is used for proofing the metabolites and also to measure them and the metabolites are moved into it after GC separation (Jasim et al., 2015). For the particles to have an additional division on the basis of m/z proportion, they need to be ionized, so that they can be identified and changed into an electronic signal. Following ionization, fragments are divided into mass analysers and identification is done. Every particle produces its own mass spectrum (Allied Academies Conference, 2019). This is then analysed against available mass libraries in the form of stored mass spectrum. This is accompanied by retention index or retention time comparing with chromatograms. Once, this is done, data is treated and processed, univariate or multivariated analysis and later validated (Sugimoto et al., 2012).

Clinical field requires an ideal mix of chromatographic procedures coupled with MS utilization. To evaluate and detect metabolites which are in the samples for effective accomplishment of clinical metabolomics, the integration of proficient detachment processes with high reactivity and resolution MS (Allied Academies Conference, 2019).

Section B

1. To quantify proteins in medical research, biological systems and to expand clinical applications, methods for targeted proteomics have been used. To differentiate proteins of broad scales, high delicacy and high selectivity, a procedure involving mass spectrometry is used (Shi et al., 2016). Multiple reaction monitoring (MRM) or Selected reaction monitoring (SRM) is the most common method which is performed on a triple quadruple mass spectrometer (QqQ MS). This is also used to measure cell signals and detect diseases that are connected with proteins (Ronsein et al., 2015).

To quantify protein in absolute terms, SRM is a commonly used method. By characterizing a mark group of fragmented coordinate peptides, SRM “assays” are created. SRM assays consists of many applicable transitions which are tangible predecessor ion product ion pair. These SRM assays are useful for targeted peptide identification, characterization and quantification of the targeted peptide (Ronsein et al., 2015). The sample is mixed with stable isotope-labelled peptides for achieving absolute quantification of the targeted peptide. The SRM technique helps analyse 50 to 100 proteins simultaneously and it gives excellent robustness, sensitivity and specific quantification of the targeted protein (Shi et al., 2016). Depending on the physiochemical properties of peptides, some of them are isolated, ionized and identified more than the others while they are analysed in MS (Ronsein et al., 2015). When the peptides are recognized, it should be unique to the targeted protein and should not show any evidence of any missed spliced sites or amino acids. Identification of proteotypic peptides occur late and with the help of few prediction strategies using data from discovery-based experiments (Proteomics, 2019). Once the resulting samples are obtained through SRM, they can be reproduced and sent across clinical centres (Shi et al., 2016). MRM is also used nowadays due to its accuracy and continuous evaluation of virtual changes in the quantity of the virtual changes of the targeted analytes. This can be found crosswise over models instead of exactly identifying the concentration in the sample (Proteomics, 2019).

Although SRM is the best method for mass spectrometry-based innovation for targeted proteome analysis, new methods are being developed. One such is Parallel reaction monitoring technology wherein transitions are observed parallel in a solitary analysis (Shi et al., 2016). Another such method is SWATH wherein mass complex spectra created by data which is independently procured are examined for the presence of particular peptides comparing them with libraries of qualified peptide fragment spectra (Proteomics, 2019). New software and technologies need to be developed for improving these technologies for the scientists (Ronsein et al., 2015).

2. There are several types of mass spectrometers. Most common among them is Triple Quadrupole (QQQ) mass spectrometers which is one of the best models for utilizing analysers in arrangements. Although this technique has been in vogue for the past 30 years for analysing small molecules, triple quadrupole for targeted proteomics has evolved from that and made it more useful and efficient (Masiá et al., 2013).

QQQ-MS consists of three quadrupoles analysers used one after another. The first analyzer is Q1 wherein filtered peptide ions (precursor ions) of a selected m/z ratio enter (Doerr, 2013). Q1 then functions as an analyzer as the particles enter the second quadrupole Q2, wherein selected ions (collision cell) from Q1 are broken down and the resulting fragmented particles are ultimately scanned or fixed to screen a specific ion by the last quadrupole Q3 (Masiá et al., 2013). This results in an increased intensity against retention time. This applies to each predecessor ion product ion pair. To ascertain which scan mode to apply, a combination of Q1 and Q3 is used. Although there are several scan modes, Selected Reaction Monitoring (SRM) or Multiple Reaction Monitoring (MRM) is one of the more commonly used which are used for scanning quantifying compounds in a mixture (Doerr, 2013). Q1 is static so that it allows filtering of only a specific m/z ratio of predecessors through the quadrupole, while Q2 allows for collision induced dissociations and all fragments reach Q3 which is also static to permit the filtering of the resulting ions of a specific m/z ratio to pass through the quadrupole. This allows to observe precise fragment ions derived from a known mass compound and also permits for a live observation of a single known compound (Masiá et al., 2013). One important fact to be careful about is that, despite the ability to identify effectively the m/z values of an interested compound for Q1, it is pertinent that the m/z values of the produced particles are identified before structuring the SRM experiment. This can be resolved by scanning the product ions of the composite of interest and record the m/z of all the particles (Doerr, 2013).

It is definite that new technologies will be developed along with improvement in current ones. These kind of development in the field of mass spectrometry will always be seen and due to the nature of complexity in formulating the wide range of techniques and methods for finding newer methods and formula are always going to be developed (Masiá et al., 2013).

Tasks:

Section A

1. Describe the sample collection and preparation procedures before injection into a gas-chromatograph column .

2. Describe the operation of gas-chromatograph mass spectrometry.

Section B

1. Describe the application of targeted proteomics and it use of SRM mass spectrometry.

2. Describe the operation of a triple quad (QqQ) mass spectrometer in SRM mode. I add a file for the answer from my friend and the answer is correct it can help you when u answer u can choose from the reference in the answer file

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