Editorial Feature

The Role of Mass Spectrometry in Protein Analysis

Mass spectrometry (MS) is a widely used method for studying protein complexes and biological processes.

The Role of Mass Spectrometry in Protein Analysis

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In MS-based protein identification, proteins are digested into peptides, separated, fragmented, ionized, and analyzed by mass spectrometers. Computer algorithms identify proteins from the mass spectra peaks, each representing a peptide fragment ion.

Understanding Mass Spectrometry

The primary goal of mass spectrometry is to measure a molecule's mass, extracting structural information. The process begins with ionization, where molecules transition from the solution phase to the gas phase. The ions are then concentrated into a beam and sent into a collision cell, where they dissociate. Mass analysis determines the masses of the ions or their fragments using the mass-to-charge ratio (m/z).1

Key steps in MS include ionizing molecules and separating ions of different masses. Two primary techniques for ionizing biomolecules are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). ESI applies high electric currents to aqueous protein solutions, ejecting liquid droplets into the gas phase and building up charge on the protein's surface. MALDI uses laser pulses to ionize protein samples in a fabricated matrix.

ESI is preferred for analyzing complex protein samples due to its gradual ionization process, which preserves non-covalent interactions. Various mass analyzers, such as quadrupole, time-of-flight MS, ion traps, and Fourier transform ion cyclotrons, can separate ions of different masses, each offering unique advantages. These analyzers can be used separately or in tandem to create hybrid instruments, enhancing MS capabilities.

Applications of MS in Protein Analysis

The MS proteomic workflow starts with sample handling (collection, processing, and storage) and sample preparation (protein separation, pretreatment, and purification). The data processing phase includes protein identification through de novo peptide sequencing, spectrum library search, and database search.2

Several MS workflows have been tailored for protein complex research, including:

  • Absolute Quantification of Ubiquitination (AQUA) Strategy: This approach involves creating synthetic heavy peptides representing ubiquitination linkages. These peptides are mixed with known concentrations of sample peptides and analyzed by MS. During chromatography, the retention of heavy and light peptides converges to the same spectral peak, allowing separate selection and analysis by MS.
  • Labeling of Protein Extracts: Proteins from various cell lines are differentially labeled with distinct isobaric tags. After breakdown, the peptides from each sample are combined and analyzed by MS, with each sample's peptide falling under the same m/z peak. 
  • Label-Free Quantification: Samples are examined independently and compared. A cell lysate extracts the desired protein complex using affinity purification. After digestion, the complex is examined using MS to determine its components and any alterations, with relative quantification achieved by comparing precursor ion counts or intensities. 
  • Crosslinking: Crosslinkers form covalent bonds between two amino acids in target proteins. After digestion, the crosslinked materials are converted into peptides, with some retaining the crosslink. Non-crosslinked peptides are eliminated in an enrichment phase. MS determines the crosslink site, allowing selective peak selection and fragmentation. 
  • Native MS: Isolated and purified whole protein complexes are analyzed using MS. The intact mass indicates the sample's stability, homogeneity, and subunit stoichiometry. Different associated proteins or subunits separate after peak isolation and MS fragmentation, enabling their independent identification. 

Identifying proteins accurately from tandem mass spectra is highly challenging. Current techniques typically identify less than 50 % of the proteins in complex samples.3 Consequently, new techniques are continuously developed to enhance identification accuracy and reliability. 

Future Outlook 

Mass spectrometry methods can be employed in any field of biological study, both basic and clinical, for qualitative and quantitative peptide and protein bioanalysis, posttranslational modifications, and protein-protein interactions. This technique can be used in various biological materials, both normal and diseased.4

The widespread use of automated MS machines and clinical applications of MS can enhance clinical diagnostic decisions by providing faster, simpler, and more accurate results. MS-based testing procedures offer better precision than conventional assays. Additionally, advancements in MS techniques support the growth of new disciplines by enabling a switch from focused to non-targeted proteomic approaches.

More from AZoOptics: Exploring Advancements in Cryo-Electron Microscopy for Single-Particle Analysis

References and Further Reading

  1. Olshina, MA., Sharon, M. (2016). Mass Spectrometry: A Technique of Many Faces. Q Rev Biophys. doi.org/10.1017/S0033583516000160
  2. Neagu, AN., et al. (2022). Applications of Tandem Mass Spectrometry (MS/MS) in Protein Analysis for Biomedical Research. Molecules. doi.org/10.3390/molecules27082411
  3. Wang, P., Wilson, SR. (2013). Mass spectrometry-based protein identification by integrating de novo sequencing with database searching. BMC Bioinformatics. doi.org/10.1186/1471-2105-14-S2-S24
  4. Xiao, GG., Recker, RR., Deng, HW. (2008). Recent advances in proteomics and cancer biomarker discovery. Clin Med Oncol. doi.org/10.4137/cmo.s539

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Written by

Ilamaran Sivarajah

Ilamaran Sivarajah is an experimental atomic/molecular/optical physicist by training who works at the interface of quantum technology and business development.

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