ABSTRACT
In this case, fluorogenic reagents that generate fluorescence after derivatization without fluorescence themselves are strongly recommended because of low background fluorescence affording highly sensitive detection of the derivatives. A few fluorogenic reagents for amines can be taken for the above use: orthophthalaldehyde [1] and fluorescamine [2]. As to the derivatization of cysteine residues in proteins, thiol specific reagents are requisites. Ammonium-7fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) [3] and 7-chloroN-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) [4] are most appropriate among the existing reagents for the following reasons. First, their relatively small molecules enable them to react completely with cysteine residues in proteins under a mild condition. Second, derivatized proteins are hydrophilic and thus do not precipitate after the complete derivatization. Third, their derivatives are highly fluorescent with longer emission wavelength than natural fluorescence of biomolecule (less than 400 nm). A chromatographic technique, high-performance liquid chromatography (HPLC) is also a powerful tool for separation of compounds in a complex matrix of bio-samples. However, it has not been usually used for protein separation in bio-samples except for the recently developed FD-LC-MS/MS method [3-6] for proteomics analysis. It consists of fluorogenic derivatization (FD) of proteins using fluorogenic reagent such as SBD-F or DAABD-Cl, followed by HPLC separation, detection and quantification of the derivatized proteins, isolation of the subject proteins, enzymatic digestion of the isolated proteins, and identification of the proteins utilizing HPLC and tandem MS with a database-searching algorithm. The schematic diagram of the FD-LC-MS/MS method is shown in Fig. 1.1. First, a proteins mixture is derivatized with a fluorogenic reagent and analyzed by HPLC-fluorescence detection. Next, a derivatized subject protein can be isolated without losing any amino acid sequence information including protein isoforms and posttranslational modification, and then digested by trypsin. Finally, the obtained peptides mixture is subjected to HPLC, which is connected to electrospray ionization (ESI)-MS/MS, to identify the isolated protein using the probability-based protein identification algorithm. The derivatized proteins are detected and quantified at the femtomol level [3, 4]. The quantification is performed by calculating the peak intensity of the derivatized protein that corresponds to the amount
of the derivatized protein existed. The accuracy of the method was acquired based on the reproducibility of the peak heights using the three representatives of the high, medium, and low peaks obtained from each individual mouse liver sample [7]: The relative standard deviation (RSD,%) for each between-day peak was less than 16 (a relatively high peak), 17 (medium peak), and 23% (a relatively low peak) (n = 3). The reproducibility of the retention time was also calculated using the relatively low peak, resulting in the between day RSD of 0.41% (n = 3). A remarkable feature of the method is the use of the fluorescence for the sensitive detection [3, 4] and of HPLC for the reproducible quantitation of the derivatized proteins [7] as mentioned above. It also requires only simple apparatus, consisting of a pump, a column, and a fluorescence detector. Currently, a comprehensive profiling analysis in HPLC-fluorescence detection needs a 10 h operation. However, in case the elution time of a subject protein is determined, it would be possible to reduce the time required for an arbitrary analysis on the subject protein by re-optimizing the separation conditions. It would also be possible to reduce the overall analysis time by adopting a higher-performance column, such as a recently available non-porous reversed phase column. Moreover, any pre-treatments of the sample are not required in the procedure of the method since only fluorogenic derivatized proteins are detected without detection of non-fluorescent compounds on LC-chromatogram. In contrast, other proteomics analytical methods, such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS: for example, ICAT and iTRAQ), require certain pre-treatments steps (precipitation, labeling, clean-up with column and enzymatic protein digestion: reviewed in [8]). In 2D-PAGE methods, pre-treatment procedure (precipitation etc.) for the sample is essential to obtain clear 2D electropherograms, because the composition of the sample is greatly influential to the resolution of every protein in electrophoresis. In case of ICAT and iTRAQ, each sample pre-treatment procedure is required since it influences the numbers of peptides observed by LC-MS analysis. However, such pre-treatment procedures would lower the sensitivity and reproducibility of the method owing to the loss of proteins existed in the sample during pre-treatment steps.
