Protein Mass Spectrometry
Protein mass spectrometry is a widely used technique for analyzing biological samples in various fields, including biomarker discovery, proteomics research, and clinical applications. Compared to other methods employed for large-scale protein characterization, mass spectrometry has become a leading tool in proteomics due to its ability to handle complex analyses. This technique is utilized to quantitatively identify and characterize proteins based on their structure, post-translational modifications, and interactions. Protein identification often involves the chemical or enzymatic digestion of proteins into smaller peptides, which are then analyzed by mass spectrometry and identified through computational methods or sequencing. Post-translational modifications are detected by observing changes in the mass of amino acid residues, with the modification sites mapped using sequencing or computational techniques. In glycan analysis and profiling, enzymatic or chemical methods are used to release glycan moieties from glycoproteins, followed by their derivatization for subsequent mass spectrometry analysis. Protein interactions can be studied by affinity co-purification, isolating a specific target protein and its interacting partners, or through global analysis using techniques like size exclusion or ion exchange chromatography before mass spectrometry analysis,
Overview
Quantitative Proteomics
Quantitative proteomics involves labeling proteins or peptides with stable isotopes for relative and absolute quantification. Chemical labeling techniques such as tandem mass tagging (TMT) and iTRAQ, or metabolic labeling using stable isotope labeling by amino acids in cell culture (SILAC), enable comparison of heavy and light isotopes for relative quantification. The mass spectrometry peak intensities from labeled peptides or proteins are correlated to their abundances. For absolute quantification, isotopically labeled synthetic peptide or protein standards can be spiked into samples for selected reaction monitoring (SRM) analysis, providing precise quantification.
Protein Mass Spectrometry
In protein mass spectrometry, protein and peptide masses are determined by measuring the m/z (mass-to-charge) ratio of their gas-phase ions. The process begins by ionizing proteins using an ion source, followed by separation of the ions based on their m/z ratio in a mass analyzer. The number of ions at each m/z value is recorded by a detector. Common ionization techniques include MALDI and electrospray ionization (ESI), both of which are used to analyze peptides and proteins by mass spectrometry.
MALDI-TOF Mass Spectrometry
MALDI (Matrix-Assisted Laser Desorption/Ionization) is an ionization method used in mass spectrometry that minimizes protein fragmentation. The sample is mixed with a matrix material that absorbs laser energy. A pulsed laser irradiates the sample, causing ablation and desorption of both the sample and the matrix, resulting in ionization of analyte molecules by protonation or deprotonation. The ions are then analyzed by mass spectrometry, enabling precise mass determination of proteins and peptides.
Electrospray Ionization Mass Spectrometry
Electrospray ionization (ESI) is an ionization technique that applies a high voltage to a liquid sample, creating an aerosol of charged droplets that ionize peptides and proteins with minimal fragmentation. ESI is particularly useful when combined with liquid chromatography (LC-MS), as the eluate from the liquid chromatography column can be directly introduced to the electrospray source for tandem mass spectrometry analysis, facilitating high-throughput proteomic analysis.