Explanation to Protein Mass Spectrometry Identification Technique

Principles and Applications: Mass spectrometry has the characteristics of high sensitivity, high accuracy, and the ability to accurately and rapidly identify proteins. Traditional mass spectrometry techniques are limited to analysis of small analytes. With the advent and development of new ionization techniques such as Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and Electrospray Ionization Mass Spectrometry (ESI-MS), convenient conditions are created for accurate and rapid identification of macro-molecules such as proteins.Currently, the enzymatic digestion of proteins, the separation of peptides by liquid chromatography, the analysis of peptide amino acid sequences by tandem mass spectrometry, and the combination of mass spectral data analysis have become the preferred protocols for the identification of proteins. This article mainly discusses the principles and applications of protein mass spectrum identification.


The basic principle of MALDI-TOF MS is to disperse analytes in matrix molecules and form crystals. When lasers are used to irradiate the crystals, the energy absorbed by matrix molecules through radiation leads energy to accumulate and rapidly generates heat, thereby sublimating the matrix crystals. This causes the matrix and analytes to expand and enter the gas phase. The mass spectra generated by MALDI are mostly singly-charged ions, so there is a one-to-one correspondence between the mass of ions in the mass spectrum and the mass of peptides and proteins.MALDI-generated ions are usually detected by a time-of-flight (TOF) detector. Theoretically, there is no upper limit to the mass of the molecules that the TOF detector can detect, as long as the length of the flight tube is sufficient. Research on biological macromolecules such as peptides.Therefore, MALDI-TOF mass spectrometry is suitable for the study of proteins, peptides and other biomacromolecules.

Features of MALDI-TOF-MS Analysis Technology

  • MALDI-TOF identification is convenient, fast, and can do hundreds of spots at the same time
  • Mainly used in identification of pure proteins or simple samples, such as 2DE spots
  • Lower cost

Sample Requirements

  • Protein solution: purity>90%, total protein>5 ug, concentration>0.1 ug/ul
  • Two-dimensional gel electrophoresis spot: coomassie blue stained spots and silver stained spots are clearly visible
  • SDS-PAGE strips: coomassie blue stained bands and silver stained bands are clearly visible



Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized.Electrospray ionization is characterized by the generation of highly charged ions rather than fragment ions, which reduces the mass-to-charge ratio to a range that most mass spectrometry instruments can detect, thus greatly expanding the molecular weight analysis range. The true molecular mass of ions can also be calculated based on the charge ratio and charge number.

Technical Features of ESI-MS

  • High throughput: tens to hundreds of proteins can be identified at one time
  • High sensitivity: samples with very low concentration can detected
  • Versatility: analysis of protein bands, co-immunoprecipitation eluates, tissue extracts, whole cell lysates, subcellular fractions and other samples

Sample Requirements

  • Protein solution: Total protein >5 ug, concentration >0.1 ug/ul. The buffer contains no detergent NP40, Triton X-100, etc.
  • SDS-PAGE strips: the coomassie blue stained strips and silver stained strips are clearly visible.

Notes for Preparation of Mass Spectrometry Samples :

  • The sample preparation process should avoid various types of contamination and repeated freezing and thawing
  • Avoid keratin contamination while preparing and cutting gel. Use dedicated gel reagents and a clean one-time cutting tool
  • For protein coomassie blue staining, the amount visible to naked eyes can generally be identified by mass spectrometry
  • Silver-stained proteins cannot use glutaraldehyde as a fixative, because irreversible cross-linking of proteins caused by glutaraldehyde can affect the result of mass spectrometry identification

Applications of Mass Spectrometry

Protein Purity Analysis and Protein Mass Determination

MALDI-TOF is a soft ionization technique that can be used to detect the presence or absence of a target protein or polypeptide, the presence of dimers, and a general assessment of purity. MALDI-TOF identifies a wide range of molecular weight. It can identify 700-60000Da protein molecular weightwith accuracy as high as 0.1% to 0.01%, which is far higher than the current routine SDS electrophoresis and high performance gel chromatography technology.

Peptide Mass Fingerprinting (PMF)

PMF is a mass map of peptide fragments obtained after proteolysis of the protein by a specific cleavage site.Since the amino acid sequence(primary structure) of each protein is different, when the protein is hydrolyzed, the resulting peptide fragment sequences are also different. Therefore, the peptide mass fingerprinting is also characteristic.The identity of the protein was identified by comparing the peptide map with the protein theoretical spectrogram in the database. Therefore, the accuracy of molecular massis a key indicator of PMF, but the post translationalmodificationof proteins may make the mass of PMF inconsistent with the theoretical value, and thus have an impact on the accuracy of protein identification.

MS/MS TandemMass Spectrometry Sequencing

Tandem mass spectrometry (MS/MS) is currently the most commonly used mass spectrometric identification method. MS/MS first determines peptide masses by mass spectrometry, and then selects high-abundance peptides for secondary mass spectrometry analysis. Peptides in secondary mass spectrometry collide with each other to cause breakage of amino acid bonds, producing peptide fragment ions that are detected by detectors. Thus, the amino acid sequenceinformation of the screening peptide is obtained. MS/MS makes use of the accurate mass information of peptide fragment enrichment, which is more specific and reliable and can greatly increases the reliability of database retrieval.

Author Bio

Prime Jones is a senior researcher from MtoZ Biolabs. She is specialized in the field of proteomics study.

Scientist, Mass Spectrometry & Metabolism @ Elucidata | BioChem Adda Exclusive

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