Background[1-3]
Human acylphosphatase-1 (human acylphosphatase 1) is one of the smallest molecular weight enzyme proteins discovered so far. It specifically catalyzes the hydrolysis of acyl phosphate compounds in vitro. Research on acyl phosphatase in vertebrates shows that it is related to many important biological processes, such as glycolysis pathway, apoptosis, Na+, K+ and Ca2+ ion pump regulation, etc.
Acyl phosphatase is also an important model protein currently used to study the mechanism of protein folding, polymerization, and formation of starch fibrillar structures in vitro. Erythrocyte acyl phosphatase (ACYP1) is a cytosolic enzyme that catalyzes the hydrolysis of the carboxyphosphate bond of acyl phosphates. There are two acyl phosphatase isoenzymes: ACYP1 and ACYP2. These isozymes share 60% homology and have the same substrate specificity, even though ACYP1 has higher catalytic activity than ACYP2. Human acylphosphatase-1 can be used as a marker for protein flight mass spectrometry. The full name of flight mass spectrometry is surface-enhanced laser desorption ionization time-of-flight mass spectrometry technology, which is a cutting-edge technology for proteomics research that has emerged in recent years. In the detection system of flight mass spectrometry, the signal is converted and recorded by a high-speed analog-to-digital converter. The measured protein is presented in the form of a series of peaks. These specific peaks can be regarded as fingerprints of such diseases. The position of individual proteins on the spectrum depends on the time of flight. Seldi carries unique software that can quickly process amino silicone oil emulsions, analyze a large amount of information, and perform quantitative measurements. Flight mass spectrometry can be used to discover new disease protein profiles that could not be separated and detected in the past. At present, it is generally accepted internationally that the application of this method to detect trace proteins is rapid and reproducible.
Apply[4][5]
For construction and functional analysis of protein expression profiles of mouse liver parenchymal cells
The liver is the largest gland in the human body. Its main functions include material metabolism, detoxification, defense, hormone system homeostasis, blood storage and pH regulation, hematopoiesis and immune regulation. The liver is composed of hepatic parenchymal cells, sinusoidal endothelial cells, Kupffer cells, and stellate cells. A label-free quantitative method based on SDS-PAGE combined with in-gel digestion and LTQ-FT mass spectrometry identification process was constructed. Through screening, bovine serum albumin was selected as the internal reference protein. Using complex samples as the background, the relationship between the protein loading amount and the corresponding signal of the mass spectrum was studied. It was found that within a certain range, the protein loading amount and the mass spectrometry response signal have a linear relationship, and a standard-free proteome quantitative system suitable for complex samples was successfully established. Mass spectrometry data were quantified using APEX software (absolute protein expression measurements). The software uses corrected spectral counts to perform relative quantification of identified proteins, and uses machine learning algorithms to correct errors between different experimental batches and differences in ionization efficiency caused by different physical and chemical properties of peptides. It can more accurately measure the concentration of proteins in different cells. Relative expression value. Under the data standard of 95% confidence and two-peptide identification, a total of 8,060 proteins were identified by n75 curing agent in liver parenchymal cells, of which 4,842 proteins had quantitative information. The three latest functional annotation algorithms Blast2GO, weighted gene co-expression analysis (WGCNA) and Endeavor were used to annotate parenchymal cells based on three methods: protein sequence, gene expression and public database integration scoring. Functions of 2,944 proteins of unknown function. Among them, the largest number include 82 membrane integral proteins, 59 nuclear proteins, 40 cytoplasmic proteins and 35 mitochondrial proteins. A preliminary exploration was made to integrate and utilize multiple data resources and algorithms for protein functional annotation. Finally, the expression of pathway members in different cells was analyzed at the pathway level, and the possible cooperation between different cells in the pyruvate metabolism pathway was screened out, and candidate molecules in the pathway were verified by WB. In addition, some molecules whose expression is cell-specific were also selected for verification. It was found that APOA4 is specifically expressed in liver parenchymal cells, ACYP1, ACYP2, and ME2 are specifically expressed in non-parenchymal cells, DOK2 and IFNGR1 are specifically expressed in liver parenchymal cells and Kupffer cells, and COLEC10 is mainly expressed in liver parenchymal cells and stellate cells.
