Editorials
Usefulness and Limitation of Protein Mass Analysis
2015 Volume 79 Issue 12 Pages 2549-2550
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2015 Volume 79 Issue 12 Pages 2549-2550
Organisms show various adaptive responses to external stimuli to maintain homeostasis. Failure of this adaptation leads to disease. To elucidate the molecular mechanism of the adaptive response not only leads to understanding of disease states, but also to the development of therapeutics. As these means, comprehensive analyses of the gene expression level, the protein expression level, and amount of metabolites have been developed. Comprehensive analysis of gene expression was first introduced from around 2000. By binding the cDNA taken from tissues or cells to glass-attached DNA called microarray analysis, it became possible to analyze over 20,000 genes’ expression at once. Many gene expression analyses have been performed in all under stimulation of any organ. Some of these have been lead to the detection of a diagnostic marker or drug discovery target. In recent years, because the genome information has been enhanced, gene expression analysis by mRNA sequence has become mainstream in gene expression analysis. mRNA sequence is a great tool for quantitatively evaluating the expression of a very small amount of mRNA. In addition, mRNA sequence enables us to evaluate the relative amount of expression among homolog genes and splicing variants.
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On the other hand, comprehensive and quantitative evaluation of the protein expression level has been difficult compared with gene expression analysis. However, protein expression analysis is becoming available using mass spectrometry. Comprehensive protein expression analysis using mass spectrometry is performed as follows. First, the proteins are extracted from cells or tissues and digested with trypsin or other proteases. The resulting peptides are separated with high resolution by applying them to the separation column, which is usually nano size. Peptides eluted from the separation column are ionized by a high-voltage electronic field and sent sequentially to mass spectrometry. A mass spectrometer has 3 main parts (Figure). The first process separates the peptides by their molecular mass using a quadrupole. Isolated peptides are degraded to ionized peptides in the next quadrupole after colliding with the noble gases. These degraded peptides are more precisely mass measured by the final mass spectrometry measurement. With these steps we can simultaneously measure the parent mass and the collision mass. Because the degradation site of the amino acids for the each protease is fixed, all proteins have their own degradation pattern known as the “finger print”. Therefore, even if the sample contains many different proteins we can individually identify the specific proteins. Recently, 3rd-generation mass spectrometry has been developed and the speed and sensitivity of peptide mass measurement have dramatically increased. We can detect over 1,000 different proteins with 1 application of a crude protein sample. We can also roughly determine the amount of each protein by the number of spectra that are assigned during the analysis. If you desire to more accurately quantify the specific proteins, you can use triple quadrupole mass spectrometry. By programing the specific transient pattern of each protein you can perform accurate and quantitative evaluations of approximately 100 different types of proteins in the mixed crude sample at once. The sensitivity of quantification of proteins using triple quadrupole is almost comparable to that of Western blotting, but the analysis itself has a much higher throughput. When multiple evaluations of the amounts of proteins in many samples are necessary, quantitative multiple mass analysis would be a strong tool. Also, by using standard quantified labeled peptides, absolute quantification of protein amounts is possible. Relative quantification of protein samples is used for the most comprehensive analysis of protein amount. For that purpose, radioactive labeling of the whole protein content of the cell or post-extraction labeling by crosslink chemicals such as iTRAQ are used. Using these methods we can evaluate the different amounts of 1,000 or more proteins in crude samples.1,2
Structure of tandem mass spectrometer: (A) nano-liquid chromatography; (B) first separation mass spectrometry; (C) collision cell; (D) final separation mass spectrometry.
Comprehensive quantitative evaluation of the amount of protein gives valuable information that cannot be obtained by the evaluation of the level of expression of mRNA; for example, information on the degradation or transport of proteins. Specifically, determination of marker genes contained in the blood cannot be performed other than by mass spectrometry.3
But many challenges still remain with protein analysis using mass spectrometry compared with gene expression analysis. The diversity of proteins is high for all the procedures, including extraction from tissue or cells, degradation by protease and separation by column. The difficulties with handling peptides do not exist when fragments of DNA are handled. Small molecular weight or hydrophobic proteins are sometimes not even identified by mass peak. In particular, the relative quantitative evaluation of different types of proteins is extremely difficult. Although gene expression quantification can detect several hundred molecules, the detection limit of mass protein analysis is several amole. Contamination of major contained protein reduces the sensitivity for the detection of target proteins. Also, the mass spectrometry system itself is very expensive, and the operation, including the nano liquid chromatography, requires great skill. Marked increases in the speed and sensitivity of mass spectrometry would not be expected after the debut of 3rd-generation mass spectrometry. The sensitivity of the current mass analysis almost reaches the limit of the detection and further increases in sensitivity would not be expected unless a method of protein amplification is developed. Importantly, the sensitivity of protein identification rather depends on the method of preparation of the proteins before being applied to mass analysis. Principally, the method of protein preparation largely changes the sensitivity of mass analysis. Even one major contaminant (eg, albumin in serum or myosin in myocardial tissue) attenuates the detection sensitivity for small amounts of other proteins. Efficient removal of major contaminating proteins and specific purification of the target proteins are essential to obtain accurate and highly sensitive mass results. For that purpose we have to develop the most suitable pre-preparation methods for the target proteins. Novel fractionation methods of protein purification are expected to develop. In the cardiovascular field, efficient removal of myosin and mitochondrial proteins might be the key to high-sensitivity mass analysis. Regardless of these difficulties, mass spectrometry enables us to efficiently identify the specific target protein with high sensitivity. In particular, identification of binding protein after immunoprecipitation using mass spectrometry has been a very strong tool for evaluating novel signal transduction.4 For efficient usage, it is important to know the usefulness and limitation of mass analysis and to develop novel and the original methods of pre-preparation of target proteins before applying mass analysis. Mass analysis has been a strong tool for new discoveries in molecular biology and should be used routinely also in the cardiovascular research field.
