More than 300 spots were identified in the 20-PAGE gels from the T-lymphocytes of the normal and asthmatic patients. The general distribution pattern of the spots in the silver-stained gels was similar in both groups (Fig 1, top left, A, and top right, B). The spots in the area of pi 4 to 7, and molecular weight of 20 to 100 kd were analyzed by an image analysis program (ProteomWeaver). Protein spots of the normal and asthma groups were compared, and 25 proteins showed different intensity, suggesting the differential expression. Among them, the intensities of 13 spots were significantly increased and the intensities of 12 spots were decreased in the asthma group compared to the control group. The 25 selected spots of the 2D-PAGE of an asthmatic, nonsmoking, 27-year-old man are shown in Figure 1, bottom, C.
Identification of the Differentially Expressed Proteins by MALDI-TOF-MS
After destaining, extraction, and lysis with trypsin, the individual spots were identified by MALDI-TOF-MS and this was followed by a database search (MS-FIT). On the list of 20 candidate proteins for each spot, the final protein was determined by comprehensively considering the corresponding experimental isoelectric point, the molecular masses, the number of matched peptides, and the sequence coverage. Thirteen up-regulated and 12 down-regulated proteins in the asthmatic group were identified (Table 1). Testing for multiple comparisons by FDR analysis resulted in an FDR threshold (a) value of 0.0396468. Treat asthma and read more about it on My Canadaina Pharmacy – https://mycanadian-pharmacy.net/category/asthma.
PDE4C, heat shock protein 70 kd (HSP-70), protein tyrosine phosphatase (PTP), (P-arrestin-1b, thioredoxin-like 2, LLR protein, glutathione reductase, major histocompatibility complex class I antigen, epidermal growth factor receptor (EGFR), pyr-roline 5-carboxylate reductase isoform, and FLJ25770 protein were increased in the T-lympho-cytes of asthmatic patients. Dynein, cytoplasmic light intermediate polypeptide 2, vimentin, zinc finger protein 76, tubulin (P2, T-cell receptor (P-chain, cyclin-dependent kinase 6, pyridoxal kinase, phenylalanine hydroxylase, GST-M3, and tetratricopeptide repeat (TPR)-containing protein were decreased in the T-lymphocytes of the asthmatic patients.
We performed RT-PCR using PDE4C-, thiore-doxin-2-, and GST-M3-specific primers. The results confirmed that the messenger RNAs of PDE4C and thioredoxin-2 were expressed at very low levels in the blood of normal subjects, and they were present at considerably higher levels in the blood of asthmatic patients. The messenger RNA level of GST-M3 in the asthmatic patients was obviously less than that in normal subjects (Fig 2). These results substantiate the specific up-regulation of PDE4C and thiore-doxin-2 and the down-regulation of GST-M3 in the blood of asthmatic patients compared with those of the normal subjects.
Figure 1. Representative 2D-PAGE gels of the peripheral T-lymphocytes of normal patients (top left, A) and asthmatic patients (top right, B). Each image shows a similar expression pattern of spots between top left, A, and top right, B. Twenty-five selected and identified spots are shown by the numbered arrows in a 2D-PAGE gel (bottom, C) of an asthmatic, nonsmoking, 27-year-old man (solid arrows indicate increased spots of proteins; empty arrows indicate decreased spots of proteins).
Figure 2. RT-PCR analysis of the messenger RNA expressions of the differentially expressed PDE4C protein, thioredoxin-2, and GST-M3 protein. RT-PCR of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for RNA variation. 100bp = 100 base pair.
Table 1—Identification of the Proteins With Altered Expression Levels in the Human T-Lymphocytes of Asthma Patients Relative to Normal Persons
|No.||Protein Name||Expression||MS-FIT Database Access No.||Cov,
|T Mass, kd/pl||O Mass,
|6||Pyrroline 5-carboxylate reductase isoform||1||4960118||24||33/5.7||28/6.0||1.4||0.046|
|7||Unnamed protein product||35745||15||48/8.2||25/6.6||1.5||0.031|
|8||Unnamed protein product||7020470||45||26/6.0||75/5.0||1.3||0.022|
|12||Major histocompatibility class I antigen||27729531||46||31/5.6||33/5.9||1.5||0.023|
|14||Dynein, cytoplasmic, light intermediate polypeptide 2||2||19684163||21||54/5.9||59/4.2||5.1||0.010|
|16||Zinc finger protein 76||2||30583471||15||61/5.7||58/6.2||1.5||0.046|
|18||T-cell receptor 3-chain||2||3089433||27||34/6.2||32/4.2||2.8||0.038|
|22||Unnamed protein product||2||7023347||29||16/6.9||17/6.5||2.5||0.013|
|23||Cyclin-dependent kinase 6||2||51094901||23||36/6.0||35/5.9||1.5||0.022|
|25||Tetratricopeptide repeat-containing protein||2||20378333||35||20/5.0||18/5.8||1.5||0.010|