Endoproteinase Lys-C/trypsin sequential digestion and trypsin digestion were used in 293 T cell proteomics sample preparation and the results of Lys-C/trypsin sequential digestion and trypsin digestion in proteomics sample preparation was systematically evaluated. It was found that the number of identified peptides and proteins increased significantly, and missed cleavage sites, especially K sites decreased dramatically through Lys-C/trypsin sequential digestion. And the average sequence coverage of identified proteins in Lys-C/trypsin sequential digestion sample was higher than that in trypsin digestion sample. Besides, different amount of enzymes was tested to select the optimal usage of enzymes in Lys-C/trypsin sequential digestion. This study provided the references for proteomics sample preparation.

In proteomic research, to improve protein solubility of membrane proteins and nuclear proteins, buffers containing high concentration of detergent, such as 4% SDS, were widely used. However, high concentration of detergent might severely interfere with the downstream proteomic analysis, including protein quantitation and trypsin digestion. To improve the proteomic compatibility of buffers with high concentration of detergent, we used short gel method to pretreat buffers containing detergent. Protein samples were first separated by a short (2-2.5 mm) SDS-PAGE electrophoresis, and proteins were quantitated by comparing with bovine serum albumin standards via optical density analysis. The gel was then cut and peptides were recovered using in-gel digestion. The quantitative linearity range of this method was 1 to 8 μg. The quantitation was accurate and reproducible. After short gel analysis, recovered peptides generated high mass spectrometry signals. In conclusion, short gel method eliminated the interference of high concentration detergent in the proteomics analysis, and it was suitable for protein samples' pretreatment, and was worth to apply in proteomic research.
Detergents ; chemistry ; Electrophoresis, Polyacrylamide Gel ; methods ; Mass Spectrometry ; Membrane Proteins ; chemistry ; Nuclear Proteins ; chemistry ; Proteins ; chemistry ; Proteomics ; methods ; Trypsin

Objective To investigate the relationship between red blood cell distribution width (RDW) and prognosis in patients with multiple myeloma (MM).Methods The population that studied consisted of 27 patients with multiple myeloma and 30 healthy controls.The RDW was calculated according to the results of blood routine examination and compared between patients and healthy controls.Then,compared the difference between the two groups of RDW.MM patients were treated with international standard staging (ISS),and the differences of RDW in different stages were analyzed.ISS staging was used to draw the receiver operating curve (receiver operating characteristic curve,ROC curve),then take RDW14.65 ％ as the best cut-off point,the MM patients were divided into low RDW group (RDW=14.65 ％) and high RDW group (RDW ＞14.65％).Overall survival (OS) condition were compared between the above two groups.The impacts of RDW on OS were analyzed by Kaplan-Meier and Log-rank test.Results The average RDW value in experimental and controlled were 15.60 ％ ± 2.35 ％ vs 12.72 ％ ±0.61 ％ separately (t=6.201,P＜0.001),with statistical differences.The average RDW value in low ISS(Ⅰ + Ⅱ stage) and high ISS (Ⅲ stage) were 13.99 ％ ± 1.08％ vs 16.55 ％±2.39％ separately (t=3.800,P=0.001).The median survival time of low RDW and high RDW group was 13 months and 8 months respectively,and the difference was statistically significant (x2=6.481,P =0.011).Conclusion RDW increased in patients with MM,the risk stratification higher prognosis is worse.

The stable isotope labeling by amino acids in culture (SILAC) based quantitative proteomics serves as a gold standard because of the high accuracy and throughput for protein identifications and quantification. In this study, we discussed the application of SILAC technology in mammal model, and developed quantitative internal standard for comparative proteomics of disease model. The C57BL/6J mice fed by special diet containing the 13C6-Lysine and bred F2 generation. We identified and analyzed total proteins of 9 mice tissues of F2 generation, including brain, lung, heart, stomach, intestine, liver, spleen, kidney, and muscle. Quantitative analysis information could evaluate the mice and different tissues' labeling efficiency. Liver was the most efficient, brain the least, and the labeling efficiency were 96.34%±0.90% and 92.62%±1.98% respectively. The average of the labeling efficiency of F2 generation was 95.80%±0.64%, which met the international standard (≥ 95%) for SILAC quantitative proteomics effective study. SILAC technology was successfully extended to mammalian model system, which will provide powerful tools for the mechanism study of the pathophysiology process with mouse model.
Amino Acids ; chemistry ; Animals ; Diet ; veterinary ; Isotope Labeling ; Lysine ; chemistry ; Mice ; Mice, Inbred C57BL ; Proteins ; chemistry ; Proteomics ; methods

OBJECTIVE To explore the value of providing pharmaceutical service related to risdiplam in direct-to-patient （DTP） pharmacies. METHODS The follow-up data of spinal muscular atrophy （SMA） patients who purchased and used risdiplam from Shangyao Yunjiankang Yiyao Pharmacy （Shanghai） Co.， Ltd. from May 2021 to January 2023 were collected. The medication information， therapeutic efficacy and the occurrence of adverse events were retrospectively analyzed. RESULTS A total of 42 prescriptions were checked by pharmacists in the DTP pharmacies， and 7 prescriptions were found to be unreasonable （16.7%， 7/42）， which were corrected after the timely intervention. During the follow-up management， pharmacists replied to 4 patients （9.5%， 4/42） regarding medication consultation about medication requirements and adverse events. Two patients with type Ⅰ SMA experienced adverse events： one of them presented with fever and the other presented with skin dryness with darkening. Both of them were grade Ⅰ toxic reactions and generally did not require clinical treatment. Considering that the patient sustained low-grade fever for a long time， the pharmacist suggested symptomatic treatment under the guidance of the doctor. CONCLUSIONS Pharmacists in DTP pharmacies conducting follow-up management of risdiplam use for rare disease SMA patients can help promote rational， standardized medication for patients.