What Is Protein Mass Spectrometry Technology?

Background of Protein Mass Spectrometry

Protein mass spectrometry refers to the use of mass spectrometry for protein research and characterization, including protein quantitative detection, characteristic analysis, interaction mapping, and post-translational modification identification. Protein mass spectrometry, also known as mass spectrometry-based proteomics, includes three methods. The first method is to analyze undigested or intact protein samples by mass spectrometry, usually using high-resolution matrix-assisted laser desorption ionization (MALDI) mass spectrometry or electrospray ionization (ESI) orbitrap mass spectrometry (ESI-MS). The second method is to analyze the digested protein fragments by mass spectrometry. Shotgun proteomics is a branch of the second method and has been widely used. The method was used to identify proteins in complex mixtures by high performance liquid chromatography-mass spectrometry (HPLC-MS). The third method, which uses mass spectrometry to analyze larger peptides.

Fig. 1 Schematics of top-down and bottom-up proteomics analysis. (Anca-Narcisa Neagu, 2022)

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• Hybridoma Sequencing
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Working Principles of ESI-Based Protein Mass Spectrometry

The principle of protein mass spectrometer is that digested or intact protein is introduced into the sample injection system and then ionized by ion source. Among them, MALDI mass spectrometry uses soft ionization technology, that is, laser irradiation is used to irradiate the protein sample plate or matrix to vaporize the sample without producing fragment ions. However, some macromolecules may not be resistant to high temperatures and are not suitable for MALDI protein spectrometry analysis. The ion source of the ESI protein spectrum is electrospray technology.

ESI protein mass spectrometry technology uses electrospray technology to generate ion flow, which is then flowed to the mass spectrometer. Mass analyzers are usually quadrupole mass analyzers or ion trap analyzers, which form an electric field around the ion flow. The mass analyzer can attract cationic peptides with a specific mass-charge ratio and capture them. When the ion passes through the electric field, the ion detector can detect and measure the recording quality, the charge, and the time to arrive at the detector.

The original ESI mass spectrometer was equipped with only one mass analyzer and one ion detector, while today's mass spectrometers are equipped with multiple ion cells. These ion pools are usually located on both sides of the ion source and ion sensor and sandwiched between the electrodes. The ion sensor can adjust the frequency and voltage to select the desired range of mass-charge ratio and redirect the ion flow to further cleave peptide fragments. Ion traps can be used as mass analyzers and ion detectors, and they can also store ions. The ion source, mass analyzer, and ion detector are all in a vacuum.

When the peptide moves between the mass analyzer and the ion detector, it can be further cleaved by high-energy collision dissociation (HCD) or electron-transfer/higher-energy collision dissociation (EThcD). All these systems are connected to the mass spectrometry control software. Users can detect any range of data because a specific range of ions can be selected, and the mass spectra of these fragments can be recorded for data analysis.

Compared with the early instruments equipped with only one mass analyzer and one ion detector, the mass spectrometer with continuous mass analyzers and ion detectors is called tandem mass spectrometer. With the development and maturity of mass spectrometry technology, tandem mass spectrometers are equipped with more components. The early mass spectrometer was bulky, but now the desktop mass spectrometer is portable and can be easily placed in the corner of the lab.

Applications of Protein Mass Spectrometry

Because the mass spectrometer is designed for a specific purpose, the type of mass spectrometer is related to the specific application. The mass spectrometer based on ESI is especially suitable for sequence analysis, while the mass spectrometer based on MALDI is very suitable for quality analysis. The following are common applications of protein mass spectrometry:

1) Identification and verification of proteins (e.g., complete quality analysis, peptide mass fingerprinting or peptide mapping).

2) Protein quantification (e.g., isotope labeled quantitative proteomics, MALDI-based unlabeled proteomics, and unlabeled proteomics based on ESI analysis).

3) Protein structure determination (e.g., hydrogen-deuterium exchange or antibody protein de novo sequencing).

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• De Novo Biosimilar Monoclonal Antibody Sequencing
• De Novo TCR (T Cell Receptor) Sequencing
• De Novo Rodent Antibody Sequencing
• De Novo Rabbit Antibody Sequencing
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Peptide Sequence Analysis by Edman Degradation

More than half a century ago, Swedish biochemist Pehr Edman proposed a method of cyclic degradation of peptides (Edman Degradation), which is mainly divided into the following three steps:

• Cross-coupling: under alkaline conditions, phenyl isothiocyanate (PITC) combines with the free α-amino group at the N-terminal of the peptide chain or protein to form phenylthiocarbamyl (PTC) derivatives, namely PTC-peptides.
• Degradation: the N-terminal amino acid of PTC-peptide was selectively cut off by trifluoroacetic acid, and the aniline-thiazolinone (ATZ) derivative of the amino acid was released. Finally, the organic solution of the ATZ derivative was extracted from the reaction solution.
• Conversion: ATZ-derivatives are unstable and become stable phenylthiohydantoin (PTH) amino acids in an acidic solution. In this series of reaction cycles, one amputated terminal amino acid is identified in each cycle until all N-terminal amino acids are sequenced. The PTH-amino acids in each cycle are identified by chromatography or electrophoresis, and the whole peptide sequence can be inferred.

However, Edman degradation still has some limitations: 1) It is error-prone and is only suitable for sequencing short peptides (< 30–50 residues). 2) low throughput (e.g., each amino acid takes about 40min), and 3) peptides with N-terminal blockage (e.g., N-terminal acetylation) cannot be sequenced. Therefore, in order to quickly obtain the complete sequence of proteins, researchers usually use mass spectrometry.

Peptide Sequence Analysis by Mass Spectrometry

Liquid chromatography-mass spectrometry (LC-MS) has become a common method of peptide sequencing because of its ease of use and high throughput workflow. However, liquid chromatography tandem mass spectrometry (LC-MS/MS) is the preferred method for protein sequencing, which can deeply cleave proteins and produce ion fragments, thus obtaining higher accuracy and more complete information.

In LC-MS or LC-MS/MS quantitative analysis, the protease was cut into short peptide fragments. The peptides were ionized by mass spectrometry, separated according to the mass-charge ratio, and determined by an ion detector. The detector converts the charged ion strength into an electrical signal, which is processed by the data system to generate mass spectrometry. The mass spectrometry was analyzed by bioinformatics tools, and the sequence information was obtained, so as to analyze the original peptide sequence of the sample.

By analyzing the ion quality of peptide fragments of proteins, protein mass spectrometry can not only effectively identify and sequence proteins, but also be widely used in other fields, such as obtaining post-translational modification and stoichiometric information, characterizing protein structure, folding, function, and interaction, monitoring enzyme reactions, and quantitative analysis of proteins in samples.

Fig. 2 Experimental strategies to study protein post-translational modification. (Thomas E. Angel, 2012)

What Can Creative Biolabs Do?

Creative Biolabs has the world's leading protein de novo sequencing technology and is committed to the application of this technology in the field of medicine. Through this technology, the antibody proteome produced by the immune system can be comprehensively detected so as to achieve the tracking and early warning of a variety of diseases, including autoimmune diseases, cancer, and infection. At the same time, in the field of new antibody and drug research and development, Creative Biolabs also provides a new antibody discovery platform that can detect new monoclonal antibodies directly from immunized animal or human serum, which greatly shortens the cycle of new antibody development.

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