Protein identification is commonly categorized into two types: identification of individual proteins and identification of proteins in complex mixtures. The complexity of these identification processes varies. Mixed protein samples are more prevalent in practical experimental studies, presenting greater challenges in identification. This article introduces two mass spectrometry analysis techniques, peptide mapping and tandem mass spectrometry (MS/MS), tailored for the identification of single and mixed protein samples.
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Peptide Mapping Method
Peptide Mapping involves enzymatically cleaving the protein of interest into peptide fragments. The masses of these peptide fragments are then determined through mass spectrometry, and the obtained mass values are compared with theoretical masses derived from enzymatic digestion in protein databases. This process facilitates the identification of the protein.
A commonly used mass spectrometry instrument for analyzing peptide mass spectra is Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF). MALDI-TOF operates by forming a co-crystalline film through laser irradiation of the sample and matrix. The matrix absorbs energy from the laser, transferring it to biomolecules during ionization. The ions are then accelerated through a flight tube, and their time of flight to the detector is proportional to their mass-to-charge ratio (M/Z). This enables the detection of ions. Due to the production of singly charged ions in MALDI-TOF, peptide mass calculation is more straightforward, and the identified mass spectra exhibit high precision. Consequently, MALDI-TOF is widely used for obtaining peptide mass fingerprints.
Given the variability in protein sequences, it is assumed that the peptide masses produced by enzymatic digestion are unique, analogous to individual fingerprints. This uniqueness leads to the term "peptide fingerprint." Leveraging this characteristic, researchers can identify target proteins by comparing the measured peptide fingerprints from the mass spectrometer with theoretical fingerprints of all proteins in a database. As the species origin of the purified protein from a specific cell or tissue is usually known, though the protein's identity is not, theoretical protein fingerprints are generated by virtually digesting all proteins in a database belonging to the target species. Subsequently, the experimental peptide fingerprint obtained from the mass spectrometer is compared with the theoretical fingerprints in the database to identify the target protein.
The illustration below depicts a case result of protein identification through peptide mapping. In this experiment, the protein of interest underwent enzymatic cleavage by trypsin, resulting in various-sized peptide fragments. Analysis using MALDI-TOF mass spectrometry produced a peptide fingerprint (see the figure below). The fingerprint reveals a range of peptide masses, from tens to hundreds of Daltons, with each peak representing the relative abundance of a peptide fragment. Generally, peaks in the tens of Daltons indicate degradation products of peptide groups, which are typically filtered out during result analysis. To enhance precision and reduce noise, higher peaks and mass values are selected for comparison with the theoretical peptide mass spectra in the database. The corresponding proteins with a matching confidence level exceeding 95% are considered likely candidates for the target protein.
Identification of labeled site for benzodiazepine binding site on GABAARs using CGAM-Bzp by peptide mapping analysis (Staiano et al., 2017)
Limitations of Peptide Mapping Method
The accuracy of protein identification through peptide mapping is influenced by various factors. Peptide mass accuracy is not always distinguishable with high precision, especially when dealing with closely related peptide fragments. In cases where two entirely different protein sequences produce closely matched peptide spectra under the action of the same protease, the results can be vastly different. Additionally, the coupling of MALDI-TOF with two-dimensional electrophoresis, where a single gel spot may contain 2-3 proteins, poses significant challenges to precisely determining the peptide fingerprints of the target protein.
In summary, while the peptide mapping method is straightforward, employing relatively simple statistical models and providing fast analysis, the accuracy of protein identification is constrained by multiple factors. These constraints include instrument precision and interference from other proteins.
Transition to Tandem Mass Spectrometry
Peptide mapping relies on first-level mass spectrometry identification. While this method is convenient and rapid, its accuracy diminishes when identifying mixtures of multiple proteins. In current experimental scenarios, identifying unknown proteins in complex mixtures is challenging and time-consuming, and the protein may degrade during the purification process. To overcome these challenges and enhance identification accuracy, tandem mass spectrometry (MS/MS) has emerged as a valuable alternative.
The peptide mapping method, while offering simplicity and speed in analysis, encounters limitations in accurately identifying protein mixtures. In response to the complexities of unknown protein purification and the need for improved identification precision, the adoption of second-level mass spectrometry has become imperative.
Tandem Mass Spectrometry Identification
Tandem mass spectrometry (MS/MS) extends the capabilities of primary mass spectrometry by enabling the selection and analysis of a particular peak identified in the initial mass spectrum. This secondary analysis provides valuable information about the sequence of peptides present. The next step involves comparing these identified peptide sequences with the protein sequences stored in the database. Proteins with a high degree of confidence in this comparison are recognized as the target proteins.
Peptide identification in mass-spectrometry shotgun approach (Chugunova et al., 2018).
Tandem Mass Spectrometry Analysis
In tandem mass spectrometry (MS/MS) experiments, the mass spectrometer selects a peak from the primary mass spectrum, corresponding to the peptide ion at a specific mass-to-charge ratio. These selected ions undergo high-speed collisions with inert gas in the mass spectrometer, causing random cleavage of peptide bonds. Subsequently, algorithms are employed to analyze the resulting tandem mass spectrum, allowing the determination of the sequence of the collided peptide.
The process of inducing peptide bond cleavage through collisions is known as collision-induced dissociation (CID). In CID, there are numerous potential ways in which peptide bonds can break, but typically, the b-series and y-series are the primary considerations. This is because the collision voltages used in mass spectrometry experiments are generally low, making it less likely for ions from other series to be generated. The specific patterns of peptide bond cleavage play a crucial role in accurately analyzing peptide sequences. Compared to the primary mass spectrum, which only provides the mass of the peptide, the secondary mass spectrum from MS/MS can identify the sequence of the peptide, offering more precise and detailed information.
Special Considerations in Tandem Mass Spectrometry
During the high-speed collision of peptide ions with inert gas, various bonds, besides peptide bonds, may break. This can introduce some noise and interference in the subsequent analysis of the tandem mass spectrum. For example, certain acidic amino acids may lose a water molecule, or basic amino acids may lose an ammonia molecule during the collision process, impacting the analysis of peptide sequences. Additionally, post-translational modifications on amino acid residues, altering the molecular weight of the residues, can affect the determination of amino acids in mass spectrometry analysis.
References
- Staiano, Maria, et al. "Enzymes as sensors." Methods in enzymology. Vol. 589. Academic Press, 2017. 115-131.
- Chugunova, Anastasia, et al. "Mining for small translated ORFs." Journal of proteome research 17.1 (2018): 1-11.
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