3 results about "Intact protein" patented technology

The present invention belongs to the technical field of sensorineural hearing. Provided is the use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss. The use comprises: the construction of a stably transfected cell line, wherein: HEK293T cells are cultured using a DMEM culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37ºC with 95% air and 5% CO2, and when the cells reach 80%-90% confluence, cell passage is performed; IP-MS analysis, wherein: protein complexes are purified using Protein A / G immunoprecipitation magnetic beads, a portion of the extracted proteins is used as input, and then 2 μg of anti-Cx26 antibody is added to the remaining protein extract, same are gently pipetted and mixed, and incubated on a rotating shaker at 4℃ overnight; immunofluorescence observation of the co-localization of Cx26 and SPTBN1, wherein: a stably transfected cell line expressing WT-Cx26 and Mut-Cx26 is constructed; and co-immunoprecipitation (Co-IP) validation, wherein: a stably transfected cell line expressing WT-Cx26 is constructed. The present invention overcomes the limitations of therapeutic methods, such as the relatively low targeting specificity and short therapeutic time windows associated with full-length protein supplementation via gene therapy, thereby providing new insight into GJB2-related hearing loss, and a new target and a new treatment for same.

The application belongs to the field of chemical analysis, and particularly relates to a method for quantitatively analyzing intact proteins based on mass spectrometry tags. The specific technical scheme comprises the following steps: labeling a protein to be measured; separating the labeled protein by using a size exclusion chromatography-mass spectrometry combined technology; and quantitatively analyzing the protein to be measured by using a mass spectrometry mode of in-source fragmentation combined with multiple reaction monitoring. The application realizes the top-down high-sensitivity absolute quantification of intact proteins on a low-cost QqQ-MS by using a chemical labeling-in-source fragmentation-MRM combined strategy. The method for quantitatively analyzing proteins provided by the application has strong universality and good instrument compatibility.

This invention provides a mass spectrometry method for determining the 42 / 40 ratio of β-amyloid protein in human plasma, including using¹ 5 Using intact Aβ42 and Aβ40 proteins labeled with N stable isotopes as internal standards, plasma samples were extracted and purified using immunoprecipitation. Characteristic peptides were then generated through Lys-N proteolysis, and finally, targeted quantitative analysis was performed using liquid chromatography-tandem mass spectrometry. The characteristic peptide of Aβ42 is Aβ28-42 KGAIIGLMVGGVVIA; the characteristic peptide of Aβ40 is Aβ28-40 KGAIIGLMVGGVV. Compared to intact proteins, the characteristic peptides in this invention are less prone to aggregation and exhibit higher ionization efficiency in mass spectrometry, demonstrating excellent specificity, repeatability, and sensitivity, providing a reliable basis for the diagnosis of Alzheimer's disease.