Protein analysis and identification are grounded in a fundamental fact: most peptide sequences containing six or more amino acids are unique in a given proteome. Thus, by identifying a peptide sequence with at least six amino acids, we can match it to protein sequences in a database to determine the protein source of that peptide.
Mass spectrometry has emerged as one of the three core technologies in proteomics research. In the late 1980s, scientists in the United States and Japan independently developed two soft ionization techniques: matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MALDI involves embedding the sample uniformly in solid matrix crystals. When the matrix crystals absorb laser energy, it is evenly transferred to the sample, causing instantaneous vaporization and ionization without damaging the compounds. On the other hand, ESI operates under atmospheric pressure, where the analyte solution passes through a high-voltage capillary. Under an electric field of 2-6 kV, the sample solution emitted from the capillary undergoes electrostatic spraying, forming charged droplets. After a nitrogen gas curtain for drying, solvent evaporation reduces droplet size while maintaining a constant charge, leading to increased surface charge density. Eventually, the droplets rupture into charged sub-droplets due to Coulombic repulsion, repeating this process until tiny charged droplets are formed. As the droplets decrease in size, the surface electric field becomes highly potent, ultimately transforming the droplets into completely desolvated ions. In this manner, sample ions enter the gas phase in the form of single or multiple charges for mass spectrometric analysis.
MALDI-MS and ESI-MS procedures (Lin et al., 2009)
In the realm of proteomics research, protein mixtures undergo enzymatic cleavage by trypsin, yielding short peptide fragments that are subsequently separated via chromatography and introduced into a mass spectrometer. The mass spectrometer's ionization source ionizes the sample, imparting charge to the peptide segments. The relative molecular mass of these segments (MS1) is determined by recording the mass-to-charge ratio and charge state. Subsequently, the peptide segments undergo further fragmentation, and the information on fragment ions is recorded (MS2). Finally, the integration of MS1 and MS2 information is utilized for peptide sequence tag (PST) matching to identify the sequence of the peptide segment and match it to the corresponding protein. The proteomics services provided by Creative Proteomics are based on the Q-Exactive Plus instrument from Thermo Fisher, USA.
Q Exactive Plus Hybrid Quadrupole Orbitrap Mass Spectrometer
Q-Exactive is an advanced hybrid quadrupole-Orbitrap mass spectrometer developed by Thermo Fisher. Building upon the foundation of Orbitrap FTMS Exactive, it combines the high selectivity of the quadrupole ion filter with the Orbitrap high-resolution/accurate mass (HR/AM) measurement technology, resulting in further improvements in resolution, scanning speed, and sensitivity performance. The Q-Exactive mass spectrometer allows for high-throughput target or non-target screening, facilitating reliable confirmation and quantitative analysis. It serves as a versatile mass spectrometry platform for proteomic research. Similar to existing Orbitrap products, Q-Exactive's high performance, stability, and operability provide more reliable analytical results for both cutting-edge research and routine analysis.
Q-Exactive demonstrates outstanding performance in the analysis of protein expression profiles, excelling in both identification coverage and the detection of low-abundance proteins within complex backgrounds. For targeted protein quantification, the HR/AM mode significantly simplifies method development, reduces sample analysis time, and enhances data reliability. In comparison to collision-induced dissociation (CID), higher-energy collision-induced dissociation (HCD) in Q-Exactive generates superior fragments and higher-quality MS/MS spectra. The enhanced spectrum quality and broad dynamic range aid in a more in-depth exploration of the proteome, identifying additional proteins and allowing quantitative analysis of proteins not detected by other instruments.
The qualitative/quantitative analysis capabilities of the Q Exactive instrument facilitate a seamless transition from proteome discovery experiments to the quantitative analysis and confirmation of target proteins. Q Exactive LC-MS/MS is supported by a range of application software for proteomic research. Thermo Scientific Proteome Discoverer software is utilized for protein identification, modification, and various label-free differential analyses. SIEVE software is employed for non-labeled differential comparison analysis, while Pinpoint software is used to establish target protein quantification methods, conduct sample analysis, and verify results.
In the realm of proteomic analysis and identification, the chromatographic separation instrument is directly connected to the mass spectrometer's inlet at the sample outlet. The separated peptide segments are ionized by the ion source (S-lens Ion Source), acquiring a charge. Ionized peptide segments enter the quadrupole mass filter, where a specific range of molecular weights is selected, and then proceed through the C-trap into the mass analyzer (Orbitrap Mass Analyzer) for the determination of the molecular weight of the peptide segment. Subsequently, the peptide segment is transported into the high-energy collision cell (HCD cell), where it undergoes collision-induced dissociation, breaking peptide bonds and dissociating short peptides into individual amino acids or polyamino acid complexes. The dissociated amino acids or amino acid complexes are then sent back to the mass analyzer for analysis, recording different ion masses.
Tandem mass spectrometry is employed to detect the molecular masses of peptide fragments obtained in the mass spectrometry. The fragmentation of peptide segments in the mass spectrometer follows a specific pattern. The precursor ion of the peptide segment undergoes collision-induced dissociation in the collision cell of the mass spectrometer, where rapid collisions with inert gas lead to the cleavage of peptide bonds. This process forms daughter ions, generating a, b, c, x, y, and z-type series ions. The a, b, c ions retain the N-terminal of the peptide chain, with the charge remaining at the C-terminal, while the x, y, z ions retain the C-terminal of the peptide chain, with the charge at the N-terminal. Among these, b and y-type ions are more prevalent and exhibit higher abundance in the mass spectrum. The mass difference between adjacent ions in the y and b series corresponds to the mass of an amino acid residue, allowing the inference of the amino acid sequence based on complete or complementary b and y series ions.
In the process of deducing amino acid sequences, the input protein database for matching undergoes simulated trypsin hydrolysis, recording each protein's set of peptide segments and calculating the relative molecular mass of each short peptide. The short peptide's relative mass identified through MS1 is matched with short peptides in the database, selecting peptides with molecular weights close to the identified peptide segment. Consequently, the number of peptide segments used for MS2 data matching is significantly reduced. During peptide retrieval, retrieval parameters are set based on the actual usage of the sample and instrument. Retrieval parameters primarily include the database, sample species, cysteine modification form, mass error range, maximum missed cleavage sites, minimum matched peptide count, among others.
Nomenclature of b-and y-, as well as c-and z-ions generated most commonly upon collision-induced dissociation and electron capture dissociation of peptide ions (Rauniyar et al., 2007)
Tandem mass spectrometry (MS/MS) technology possesses the capability to analyze the internal structure of the target molecule, enabling the determination of the amino acid sequence for each peptide segment. By leveraging the relative molecular mass of peptide fragments detected in MS1 and the sequence information of peptide segments detected in MS2, protein identification is accomplished through retrieval from protein databases, significantly enhancing the accuracy of identification results. Currently, tandem mass spectrometry has achieved automation and high throughput in the identification process. However, given the inherent complexity and limited quantity of proteins in the proteome, it is imperative for mass spectrometric analysis techniques in proteomics to exhibit characteristics such as ultralow amounts, high throughput, and high specificity.
References
- Lin, Ying, et al. "The current state of proteomics in GI oncology." Digestive diseases and sciences 54 (2009): 431-457.
- Rauniyar, Navin, Stanley M. Stevens, and Laszlo Prokai. "Fourier transform ion cyclotron resonance mass spectrometry of covalent adducts of proteins and 4-hydroxy-2-nonenal, a reactive end-product of lipid peroxidation." Analytical and bioanalytical chemistry 389 (2007): 1421-1428.
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