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MALDI-TOF Mass Spectrometry01:19

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Mass spectrometry is a powerful characterization technique that can identify and separate a wide variety of compounds ranging from chemical to biological entities, based on their mass-to-charge ratio (m/z). The instruments that allow this detection, known as mass spectrometers, have three components: an ion source, a mass analyzer, and a detector. These spectrometers differ based on the nature of their ion source and analyzers.
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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
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Recent Progress in FD-LC-MS/MS Proteomics Method.

Hiroshi Kobayashi1,2, Kazuhiro Imai1

  • 1Laboratory of Proteomics Analysis, Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan.

Frontiers in Chemistry
|June 28, 2021
PubMed
Summary

We developed a novel proteomics analysis method, fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS), for identifying proteins in single human cells and for differential proteomics analysis. This method has successfully identified cancer biomarkers.

Keywords:
FD-LC-MS/MS methodbio-analytical chemistrydifferential proteomics analysismonolithic silica-based capillary columnnano-LC-FD-LC-mass spectrometrynano-flow LCspiderweb chromatogram

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Area of Science:

  • Bio-analytical Chemistry
  • Proteomics
  • Mass Spectrometry

Background:

  • Accurate protein identification is crucial in biological and medical research.
  • Existing proteomics methods face challenges in analyzing proteins from individual cells.
  • The need for sensitive and quantitative protein analysis methods is growing.

Purpose of the Study:

  • To introduce and detail a novel proteomics analysis method, FD-LC-MS/MS.
  • To demonstrate the application of FD-LC-MS/MS for identifying expressed proteins in individual human cells.
  • To highlight the utility of FD-LC-MS/MS in differential proteomics and biomarker discovery.

Main Methods:

  • Development of FD-LC-MS/MS, integrating fluorogenic derivatization, LC separation, and MS/MS identification.
  • Application of nano-flow LC with a phenyl-bonded monolithic silica-based capillary column for enhanced separation.
  • Utilized a probability-based protein identification algorithm for peptide mixture analysis.

Main Results:

  • Successfully identified expressed proteins in individual human cells (K562 and Saccharomyces cerevisiae) with over 1,300 protein peaks detected.
  • Demonstrated the method's capability for differential proteomics, enabling comparison of protein levels between samples.
  • Identified several cancer biomarkers using FD-LC-MS/MS.
  • Developed a novel "spiderweb" chromatogram for comparing separation profiles and conducted trials for molecular weight estimation.

Conclusions:

  • FD-LC-MS/MS is a powerful and versatile method for proteomics analysis, particularly for single-cell studies.
  • The method facilitates differential proteomics and has proven effective in identifying cancer biomarkers.
  • Advancements in LC separation and data visualization enhance the efficiency and interpretability of proteomics data.