Enrichment of phosphopeptides is an important process for mass spectrometric analysis. Here, we used the enrichment method using the biphasic Phos-tag/C18 tip, which consists of Phos-tag agarose beads overlaid on the C18 disk filter in a micropipet tip. In the cell lysates of GIST882 cells, 3187 phosphopeptides (1688 unique phosphopeptides) were identified by nano LC-MS/MS analysis coupled with phosphopeptide enrichment using this method. This method was comparable to another phosphopeptide enrichment method using Titansphere Phos-TiO Kit in numbers of peptides detected. A total number of unique phosphopeptides detected by the two methods was 3202, and the overlap between phosphopeptides enriched by two methods was 36%. Therefore, it is concluded that Phos-tag agarose method is an alternative method for identification of phosphoproteins.
アガロースゲルを一次元目に用いた二次元電気泳動法を紹介し, この方法が種々の試料にも応用できるか否かについて考察した. この方法を, 筋肉, 肝臓, 腎臓, レンズにおける全タンパク質の分離分析に用いたところ, 一次元目のゲルトップに引っ掛かることなく, ほとんどすべての成分が分析でき, 分析できる等電点, 分子量の範囲は広いものであった. 植物材料, 細胞核試料以外のほとんどすべての材料で理想的な分離が可能であった. このパターンに画像解析法を適用することにより, 骨格筋タンパク質トロポニンTやトロポミオシンの多くのアイソフォームを検出し, トロポニンTとトロポニンCの間やレンズクリスタリンサブユニットの間での蓄積の相互関係を調べ, さらにはトロポミオシンアイソフォームの組織特異的分布を調べた.
Analysis of serum γ-GTP isoenzymes was attempted with isoelectric focusing using agarose gel as a supporting medium. After testing various conditions to obtain electrophoretic patterns with good reproducibility, suspension of Ampholine in Agarose IEF was finally chosen as a supporting medium, and electrolysis at 6.25W/plate (length 12.5cm×width 11cm) for 1hr under cooling with circulating water was found to be most appropriate. A serum sample, 20μl, absorbed to a small piece of filter paper was applied onto the medium 1cm away from the cathode. Under these conditions, the pH gradient was linear in the range from pH 3.5 to 9.5, and serum proteins were clearly separated.
This method was applied to the separation of γ-GTP isoenzymes. γ-GTP was stained with γ-L-glutamyl-p-diethylaminoanilide. After electrophoresis, the gel was fixed with 50% saturated ammonium sulfate solution followed by removal of the ammonium sulfate, and then submitted to staining procedures. This pretreatment was found to give very clear and permanently preservable electrophoretic patterns. Serum γ-GTP was separeted into 7 isoenzyme bands by this method. The bands which consistently appeared were in pI range of 4.5-5.0, while a band at pI 5.5 was noted in the cases of bile stagnation, and a band at pI 6.5 was observed in cases with malignant tumors.
The cerebrospinal fluid protein fractions were investigated by the agarose isoelectric focusing (agarose IEF) and the silver staining, for the purpose of elucidating protein abnormalities in the neural tissues in the degenerative neurological diseases, especially in motor neuron disease (MND), spinocerebellar degeneration (SCD) and Parkinson disease (PD).
In this study, we focused six bands, mainly composed of transferrin, separated by agarose IEF and the silver staining. Some of these bands showed double fractions, even in the cases of the control group consisted of the patients without organic disease. In MND, all patients had double fractions of one or more of these bands, and it was interesting that in some of them double fractions were seen on the band with the isoelectric point of 5.6. Double fractions were also seen in many of SCD (69%) and PD (91%). Cases in that there was positive in more alkaline zone than transferrin in this method, were more in disease groups (especially in MND and SCD) than in the control group.
This method combined agarose IEF with the silver staining seemed to be the excellent one as the investigating procedure for cerebrospinal fluid proteins.
The electroendosmotic behavior of IsoGel and Agarose IEF was determined in a series of various wide and narrow pH range electrofocusing experiments in which glucose served as marker substance.
In IsoGel its liquid flow caused towards the anode in all wide and narrow pH range electrofocusing. The strength of segmetal liquid flow was proportional to voltage distribution across the gel. The pH gradients obtained in IsoGel when using same wide pH range ampholyte has markedly low pH values than that of Agerose IEF at acidic region.
In Agarose IEF its liquid fow behavior differed in wide and narrow pH range electrofocusing. In the case of wide pH range electrofocusing its liquid flow reversed towards the anode at pH 5.5∼6 region in the foucsed gels. The other hand, in narrow pH range electrofocusing the direction of liquid flow was differed with pH range of ampholyte. In higher pH range than Amopholine 5-7 its liquid flow coused towards the cathode and in lower pH range than Ampholine 4-6 reversed towards the anode. The direction and the streght of liquid flow in narrow pH range Agarose IEF depends on the pH distribution in the focused gel. The pH curves obtained in Agarose IEF when using same narrow pH range Ampholyte were slightly higher than that of IsoGel.