Protein Analysis FAQ

• • Q&A of Label-Based Protein Quantification

  Q1: What is label-based quantitative proteomics? A1: Label-based quantitative proteomics involves tagging proteins or peptides with chemical or isotopic labels to distinguish different samples during mass spectrometry analysis. This approach enables relative or absolute quantification of protein abundance. Common labeling methods include iTRAQ, TMT, and SILAC. Q2: What are the differences between SILAC- and iTRAQ/TMT-based protein quantification? A2: 1. SILAC is a metabolic lab

• • What Are the Commonly Used Methods for Protein Methylation Detection

  Protein methylation is a crucial post-translational modification mediated by methyltransferases, such as protein arginine methyltransferases and protein lysine methyltransferases. These enzymes catalyze the transfer of a methyl group from S-adenosylmethionine to specific amino acid residues, primarily arginine or lysine, thereby modulating protein function and activity. Accurate detection of protein methylation status is essential for both fundamental biological research and disease diagnostics. Below are..

• • CDPro and BESTSEL Yield 0% α-Helix and Low β-Content: Why Results Deviate from Literature and Can Dilution Improve Accuracy

  Using CDPro and BESTSEL to analyze circular dichroism (CD) spectra yielded an α-helix content of 0% and a markedly low β-sheet content, which significantly deviates from values reported in the literature. What recommendations can be made to improve the reliability of the experimental results? Could lowering the protein concentration (currently at 1 mg/mL) enhance measurement accuracy? When employing CD spectroscopy to assess protein secondary structure, discrepancies between calculated results and .........

• • How to Interpret Glycosylation Site Results

  The identification of glycosylation sites is typically accomplished using mass spectrometry (MS). These data allow researchers to gain a detailed understanding of protein function, its roles within biological systems, and the changes that may occur in response to genetic variations or under pathological conditions. For result interpretation, we recommend focusing on the following key aspects: 1. Location of Glycosylation Sites MS data can be used to pinpoint specific amino acid residues on the protein......

• • How to Choose a Protein Database in Proteomics

  Selecting an appropriate protein database is a crucial decision in proteomics research, as it directly influences the accuracy of mass spectrometry-based data analysis and protein identification. Below are some commonly used protein databases along with their typical applications: 1. UniProt (Universal Protein Resource) One of the most comprehensive protein databases, UniProt provides protein sequences from a wide range of species. It is suitable for proteomic studies across various organisms..........

• • How to Fix Severe Clogging and Extremely Slow Flow Rate in Sephadex G-100 Protein Purification Columns

  When using Sephadex G-100 for protein purification, issues such as severe column clogging and significantly reduced flow rate may occur. The following approaches can be considered to address these problems: 1. Examine Column Packing Ensure that the Sephadex G-100 medium is packed uniformly and free of air bubbles. The presence of air pockets or uneven packing can impair column flow and compromise separation efficiency. 2. Adjust Sample Volume and Concentration Excessive sample volume or overly .........

• • Consultation on Protein Mass Spectrometry Identification Data Analysis

  Protein mass spectrometry analysis yields various types of data, which primarily include: 1. Peptide Mass-to-Charge Ratio (m/z) Data This fundamental output of mass spectrometry reflects the mass-to-charge ratios of peptide ions. These values allow inference of the molecular weights of the peptides. 2. Peptide Fragment Ion Data In tandem mass spectrometry (MS/MS), peptides are further fragmented into smaller ions, producing characteristic fragmentation spectra. These spectra serve as the basis for peptide..

• • How Should Endogenous SUMO Co-IP Be Performed

  Endogenous Co-Immunoprecipitation (Co-IP) of SUMO (Small Ubiquitin-like Modifier)–modified proteins is typically conducted according to the following steps: 1. Cell Collection Begin by harvesting cells. For studies of SUMOylation, transiently transfected cells or cells subjected to specific treatments are commonly used to enhance the levels of SUMOylated proteins. 2. Preparation of Lysis Buffer Prepare a lysis buffer containing protease inhibitors and deSUMOylation inhibitors, such as N-ethylmaleimide (NEM)

• • How to Determine Which Amino Acids Are Present from a Mass Spectrum? How to Determine the Position of Modification

  Analysis of Mass Spectrum 1. Examination of Mass Spectral Peaks Peaks in the mass spectrum correspond to ions with distinct mass-to-charge (m/z) ratios. The masses of amino acid residues inferred from these peaks can provide insight into the specific amino acids present in the protein. 2. Calculation of Mass Differences By analyzing the mass differences between adjacent peaks, it is possible to identify individual amino acid residues, as each has a characteristic mass increment. 3. Reference to Amino Acid..

• • What Is the Principle of Determining Amino Acids by Mass Spectrometry

  The principle of amino acid determination by mass spectrometry is based on measuring the mass-to-charge ratio (m/z) of intact amino acid molecules or their fragments. The procedure typically involves the following steps: 1. Ionization The amino acid sample is first ionized to facilitate detection by mass spectrometry. Common ionization techniques include Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI). 2. Mass Analysis The resulting ions are introduced into the mass....