The total proteins were extracted (Bio-Rad) and analyzed by LC-MS (Bruker Daltonics, Germany). MS raw data were analyzed with FragPipe (v17.1) which relies on MSFragger for qualitative analysis and uses Phosopher for validation and filtering. Spectra files were searched against the homo sapiens SwissProt database (20425 entries). IonQuant mode and TMT-Integrator was used to perform isobaric labeling-based quantification (TMT/iTRAQ). Proteins denoted as contaminants were removed, the remaining identifications were used for further quantification analysis. The conditions use to filter the deferentially expressed proteins were as follows: |log2-fold change| ≥ 1.5 and adjusted P-value < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Cluster of Orthologous Groups of proteins (KOG) and Gene Ontology (GO) enrichment analyses were used to identify the significant pathways, InterPro (v5.59-91.0) was used to provide functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites, and WoLF PSORT was used to reveal the subcellular location. P < 0.05 was set as the cutoff criterion for significant enrichment.
Lc ms
Manufactured by Bruker
Sourced in Germany
The LC-MS (Liquid Chromatography-Mass Spectrometry) is an analytical instrument that combines the separation capabilities of liquid chromatography with the detection and identification capabilities of mass spectrometry. It is used to identify and quantify a wide range of chemical compounds in complex mixtures.
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4 protocols using lc ms
1
Proteomic Analysis of Human Samples
2
Synthesis and Radiosynthesis of [11C]-TKF
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The precursor of [11C]-TKF, 4-(6-(2-fluoroethoxy)quinolin-2-yl)aniline (THKF-2), was synthesized by our research group [20 (link),21 (link)]. Triflate-Ag was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Acetonitrile and ethanol of HPLC grade were obtained from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Sep-Pak tC18 solid phase extraction (SPE) cartridge (78.4 μm of particle size) and sterile filters (0.22 μm) were purchased from Waters Corporation (Milford, MA, USA).
The [11C]-TKF automated synthesis module (TRACERlab FXc) was purchased from GE medical system. Semi-preparative high-performance liquid chromatography was conducted using a Waters pump (Waters Corporation) with a Bioscan radioactivity detector. Analytical radio-HPLC (Waters Corporation) was equipped with a dual λ absorbance detector (Waters 2487), binary HPLC pump (Waters 2487) and a Bioscan radioactivity detector. NMR and LC-MS were purchased from Bruker Corporation (Karlsruhe, Germany).
The [11C]-TKF automated synthesis module (TRACERlab FXc) was purchased from GE medical system. Semi-preparative high-performance liquid chromatography was conducted using a Waters pump (Waters Corporation) with a Bioscan radioactivity detector. Analytical radio-HPLC (Waters Corporation) was equipped with a dual λ absorbance detector (Waters 2487), binary HPLC pump (Waters 2487) and a Bioscan radioactivity detector. NMR and LC-MS were purchased from Bruker Corporation (Karlsruhe, Germany).
3
LC-MS Identification of OFG Compounds
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LC-MS is an important analytical method that couples the HPLC physical separation and the mass spectrometric detection. LC-MS (Bruker, Germany) was used for an explicit detection and identification of OFG. Methanol and water 50:50 (v/v) have been used as a mobile phase and a MultoHigh 100 RP18-5μ (CS-GmbH, Germany) as an HPLC column. Moreover, OFG has been analyzed by HPLC using various detectors. These HPLC instruments were attached with a variety of detectors like UV, PDA and ECD (electrochemical detector). HPLC using UV and PDA detectors did not report detectable components in OFG possessing UV absorption. Furthermore, HPLC-ECD using various columns did not separate OFG extract components as all traces exist near the injection peak. The previous properties have been reported before for sugars.21
4
Quantification of BMV in Tape Strips
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BMV extracted from SC on the tape strips was quantified by HPLC (Shimadzu LC-2010, Buckinghamshire, UK) with UV detection (240 nm). A mobile phase of 60:40 ACN:water was pumped at a flow rate of 1 mL min−1 through a 250 × 4.6 mm HiQ Sil C18 column (Kromatek, Dunmow, UK) at 25 °C. The injection volume was 50 µL and the retention time of BMV was ~ 11.6 min; limits of quantification and detection were 0.032 and 0.01 µg mL−1, respectively. LC–MS (Bruker Daltonik GmbH, Bremen, Germany) confirmed the identity of the BMV peak and the absence of BMV metabolites in the samples.
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