Mass Spectrometry: How Is It Works - Research Paper Example

?Analysis of Galanin Using Mass Spectrometry Introduction Galanin is known to play an important role in the regulation of food intake. Poritsanos et al. (2009) report that it affects the central nervous system. They carried out studies on the relationship between obesity and the levels of galanin in the serum. It is reported that obese human beings have been observed to have high levels of serum galanin; this suggests that peripheral galanin has a role to play in the regulation of balance of energy and that high circulating galanin levels are a contributing factor to the development of obesity and obesity-related metabolic impairments. In their findings, they report that high levels of galanin in the serum can help regulate body weight, metabolic rate and carbohydrate-lipid metabolism through a mechanism that does not depend on the feeding regulation Galanin may also be responsible for high altitude induced anorexia. Singh et al. (2001) carried out studies on the roles played by galanin and neuropeptide Y in high altitudes in food uptake. Male Sprague-Drawley rats were exposed to conditions mimicking those at a high altitude of 7620 meters for 1, 7, 14 and 21 days for a six-hour period each day and to an altitude of 6096 meters for a continuous 72 hours to study the effects of intermittent and continuous exposure. Galanin and neuropeptide Y levels were estimated in various parts of the brain and plasma of exposed and unexposed rats. They found that plasma galanin levels decreased in both groups of rats. They asserted that the changes in the levels of galanin may be responsible for anorexia at high altitudes. Galanin signaling occurs through three G protein-coupled receptors. After mucosal stomach biopsies, the galanin can be analyzed using mass spectrometry. The technique Mass spectrometry involves measuring the mass of a compound, this with very high sensitivity. Mass spectrometers require molecules to be charged and in gaseous form for analysis. Peptide molecules in galanin being large and polar, are not easily transferred into the gas phase and ionised. Electrospray (ES) Fenn et al (1989) and matrix-assisted laser desorption ionization (MALDI) Karas and Hillenkamp (1988) are the ionization techniques that are used to transform the galanin into the gas-phase. Mass spectrometers measure the mass/charge ratio (m/z) of analytes. Mass spectrometry and MS/MS is applied in protein study as it makes use of the large array of genome and protein data stored in databases. The lists of peak intensities and mass-to-charge (m/z) values produced by a mass spectrometer can be processed and compared with lists generated from the theoretical digestion of a protein or the theoretical fragmentation of a peptide. Mass spectroscopy makes use of the fact that many protein molecules can be adequately displayed on a single gel. This technology was developed in the 1970s, as noted by Klose (1975) and O’Farrell (1975). Identification of the spots separated on these gels remained laborious and was limited to the most abundant proteins until the 1990s, when biological mass spectrometry had developed into a sufficiently sensitive and robust technique. In the analysis of galanin using mass spectroscopy: 1. The galanin sample will undergo vaporisation to transform it into a gaseous form. 2. The gaseous form will then be bombarded by an electron beam to generate ions. 3. The generated ions are them separated depending on their mass-to-charge ratio by an electromagnetic field in an analyser. The analyser can be Time of Flight (TOF) or a quadruple ion trap. 4. The ions are detected. 5. The ion signal is processed into mass spectra. Ionization techniques 1. Matrix-assisted laser desorption ionisation MALDI Matrix-assisted laser desorption ionisation (MALDI) employs the use of an excess of matrix material. This matrix is precipitated with the analyte molecules (the analyte contains the galanin molecules to be analysed) by placing a very small volume of the mixture onto a metal substrate and allowing it to dry. This solid is then irradiated by nitrogen laser pulses at a wavelength of 337 nm. The matrix is an organic molecule which absorbs the same wavelength as is emitted by the nitrogen lasers. Galanin, alongside many peptides, uses matrices of dihydrobenzoic acid (DHB) or ? -cyano-4-hydroxycinnamic acid. The matrices cause varying degrees of fragmentation because of the different amounts of energies that they give to galanin molecules. The ?-cyano-hydroxycinnamic acid matrix is considered to be “hotter” than DHB, and thus leads to a higher sensitivity so the latter is preferred when the ions need to be stable for milliseconds in trapping experiments rather than microseconds in time-of-flight experiments. MALDI is used for peptide analysis because: It allows for a very rapid sample analysis. It can be used with heterogeneous samples such as protein digests. It has a high sensitivity (in the femtomole range). It has a good dynamic range. It is very accurate and can generate measurements in masses below 3000 m/z. 2. Electrospray Ionisation (ESI) A liquid containing the galanin analyte is pumped at a low microliter-per-minute flow rate through a hypodermic needle at high voltage to electrospray small droplets; these droplets then evaporate and give their charge onto the galanin analyte molecules. This ionisation process is gentle and is mainly used with molecules that are polar and carry charge. Such molecules include proteins. Increasing the galanin analyte concentration brings out a linear increase in the signal strength. This increase in signal strength occurs until saturation occurs. Electrospray ionisation is important as: It is compatible with liquid chromatography. It is also compatible with tandem mass spectroscopy. It has a very high sensitivity (when used with nanoESI the sensitivity can be in the subpicomole range). It can allow for fragmentation. This is when used with tandem mass spectroscopy. It allows for multiple charging and can therefore be used to analyse proteins using limited m/z analysers. ESI-quadrupole ion traps tend to provide better resolution and accuracy compared to MALDI-TOF analysis. Sample Preparation Sample preparation involves dissolving the galanin sample in a protic volatile solvent that has a salt concentration of less that one millimole. Mass spectrometric investigations are always heavily influenced by the initial sampling procedures. For this reason, sensitivity of the whole procedure is mainly determined more by the purification strategy rather than by the sensitivity of the mass spectrometer. Galanin purification starts with a whole-cell lysate and stops with a gel-separated protein band or spot. Mass spectrometric analysis is carried out on galanin peptides obtained after enzymatic degradation of these gel-separated proteins. In principle, classical separation methods including centrifugation and liquid chromatography come before the final gel electrophoresis. When the galanin has been purified, it is advisable to minimize the number of separations. Silver-stained amounts are necessary for successful mass spectroscopic identification of the proteins (5–50 ng or 0.1–1 pmol for a 50-kilodalton (kDa) protein). Care should be taken to minimize any contamination with keratins by contact with dust and handling without gloves. The peptides in the keratin can easily displace the galanin spectrum and replace it. Dialysis of the galanin sample may be required because most salts and detergents are incompatible with mass spectroscopy. If needed, the galanin can be eluted from a reverse-phase media and then low-pressure traps be built into the mass spectrometer injection ports. This would provide the best sample preparation method. When using affinity-based techniques it is important to note that contaminating the galanin proteins will hinder analysis. For mass spectroscopic analysis, the salts and detergents are gotten rid of in the gel washing process. The protein should be as concentrated as possible in the gel so as to avoid any excessive background in the mass spectroscopic analysis. Grouping of the protein spots is not advantageous, as it will also lead to an increase in background. Silver staining, Coomassie staining and radioactive labeling are all compatible with MS analysis and should help in the visualization of the galanin proteins. Strong oxidising agents ought to be avoided because they cause interference to the extraction of the galanin peptides from the gel. According to Shevchenko et al. (1996) it may also lead to chemical modification of the peptides. Silver staining provides adequate sensitivity, but its limitation is its narrow linear range. Galanin bands are removed and put through a series of steps including reduction, alkylation, several washing steps, and enzymatic digestion. This ends with peptide extraction. Both electrospray analysis and MALDI analysis need the galanin peptides to be concentrated and desalted. This can be carried out by using several columns of reversed-phase material (~50 nl in packing volume) in nanospray needles or gel loader tips or by injection into liquid chromatography MS Columns. The galanin peptides are then eluted onto the MALDI target or into an electrospray spraying needle. For whole proteins, MALDI uses the following procedure as a guideline: A saturated solution of ?-cyano-4-hydroxycinnamic acid is prepared in water, acetonitrile and trifluoroacetic acid (TFA) in a ratio of 1:1:0.1. The solution is to be mixed thoroughly. The solution can be used when the undissolved solid settles. Matrices can be washed with a solution containing 300ul water and 0.2% TFA before the final preparation so as to remove any additional salts. 0.5ml of the protein solution is placed directly on the plate and 0.5ml of the matrix solution added. For digested proteins, such as the galanin sample, the following procedure can be used to prepare the sample: Mix 2.5?l galanin sample, 0.4?l trypsin, 1.25?l sodium hydrogen carbonate with 6?l water and digest at a temperature of 37?C overnight. Quantitative Mass Spectrometry The intensity of the mass spectrometric signal obtained cannot be correlated easily with the amount of galanin present in the sample. To measure the level of galanin present in the analyte, stable isotope methods need to be incorporated into the MS-based analysis; these can allow relative changes to be determined. The principle works by incorporating a stable isotope derivative in one of the two states to be compared. Stable isotope incorporation shifts the mass of the peptides by a predictable amount. The ratio of galanin analyte between the two states can then be accurately determined by measuring the peak ratio between the derivatised and the underivatised sample. A stable isotope that can be used is 15N. It should be noted that the sensitivity requirements for these methods are higher than for straightforward identification. The reason for this is that the peaks need to be well defined to allow for the measurements of the isotope ratios. Ironically, Gygi et al. (1999) report that the isotope label is attached during the chemical processing of the galanin proteins, causing the reactive cysteine groups in the galanin to be blocked. To overcome this, a blocking group that consists of biotin label and a linker with either 1H or 2H atoms is employed. This method is advantageous because of the fact that the label that is incorporated during galanin workup simplifies the resultant mixture because only cysteine-containing peptides will be present. Conclusion Adequate monitoring of the levels of galanin in the digestive tract can help in predicting one’s risk for developing metabolic disorders such as anorexia and obesity. By making use of mass spectrometry to determine the presence and levels of galanin, clinical research facilities and hospitals can better understand the roles played by the nervous system in the dietary system and probably aid in the development of drugs to counter these metabolic disorders. Employing mass spectroscopy though for analysis of galanin levels in the digestive tract comes with a risk of contaminating the samples (by foreign particles such as dust) to be analysed. The contamination can lead to wrong or erroneous result. This however, can be overcome by working in a clean environment and using gloves when carrying out the analysis. References Fenn JB, Mann M, Meng CK, Wong SF, 1989. Whitehouse CM. Science 246, pp.64–71. Gygi S.P., Rist B., Gerber S.A., Turecek F., Gelb M.H. and Aebersold R., 1999. Nat. Biotechnol. 17, pp. 994–999. Karas M. and Hillenkamp F., 1988. Anal. Chem. 60, pp. 2299–2301. Klose J. 1975., Humangenetik 26, pp.231–243. Matthias M.,,Ronald C.H., and Akhilesh P., 2001. Analysis of proteins and proteomes by Mass Spectrometry. Annu. Rev. Biochem. 70, pp.437–473. O’Farrell P.H., 1975. J. Biol. Chem. 250, pp.4007–4021. Poritsanos N.J., Mizuno T.M., Lautatzis M.E. and Vrontakis M., 2009. International Journal of Obesity, December, 33(12), pp. 1381-1389. Singh S.N., Vats P., Shyam R., Suri S., Kumria M.M., Ranganathan S., Sridharan K. and Selvamurthy W., 2001. Nutritional Neuroscience, 4(4), pp. 323-331. Sunia A. T., William W. and Gary S., 2002. Peptide and Protein analysis with mass spectrometry. Spectroscopy ,16, pp. 15-28. Vicki H.W, Katheryn A.R, Qingfen Z. and Guilong C., 2005. Mass spectrometry of peptides and proteins. Methods 35, pp. 211–222. Read More