Mass spectrometry (MS) serves as a powerful tool for unraveling the intricacies of proteins by transforming them into gas-phase ions and segregating them based on their mass-to-charge ratio (m/z). The integration of MS with database searches facilitates protein identification across various sample types, spanning from protein solutions to gel strips or spots. A quintessential application of this synergy is observed in the analysis of protein solutions or SDS-PAGE bands, often employing liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Principles of Protein Gel Strip Analysis by Mass Spectrometry
The fundamental principle underpinning tandem mass spectrometry (MS/MS) for protein detection involves the enzymatic digestion of proteins into peptide segments. Upon ionization within the mass spectrometer, these peptides carry specific charges. The detector meticulously analyzes these peptides, extracting their mass-to-charge ratio (m/z) to unveil their relative molecular masses. To unravel sequence information, the mass spectrometer selectively fragments certain peptides, leading to secondary mass spectrometry.
The intricate process of protein identification encompasses the analysis of mass spectrometry data through retrieval software, aligning it with pertinent databases. Evaluation is performed through scoring, with successful identification declared when the score surpasses a predefined threshold. Conversely, a failure is declared when the score falls below the threshold.
Delving deeper into the specifics of protein separation through methodologies such as two-dimensional electrophoresis (2D) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the separation of protein mixtures is achieved. Targeted protein gel spots or bands are meticulously collected, and following in-gel enzymatic digestion, peptide mixtures are extracted and identified using LC-MS/MS.
The ionized peptides, each carrying a distinctive charge, undergo detector analysis to discern their m/z values, thereby exposing their relative molecular masses. To glean sequence details, the mass spectrometer selectively fragments certain peptides, leading to secondary mass spectrometry. Retrieval software then aligns the secondary mass spectrometry data with pertinent databases, factoring in matching scores and error filtering to discern the precise sequences of the peptides. This intricate process culminates in the assembly of complete protein sequences, enabling the robust identification of proteins and opening new avenues for in-depth proteomic analysis.
Characterization of a modified protein by GeLCMS (Shevchenko et al., 2006)
Decolorization Methods:
a) Silver Staining: Gel strips are treated with a mixture of 30mM K3Fe(CN)6 and 100mM NaS2O3 in a 1:1 ratio. After the silver color fades, thorough washing is performed until the strips become colorless.
b) Coomassie Staining: Gel strips undergo treatment with a mixture of 50mM NH4HCO3 and ACN (acetonitrile) in a 1:1 ratio. The process is conducted at 4°C with repeated cycles until both the solution and gel blocks turn colorless.
Reduction, Alkylation, and Enzymatic Digestion:
a) Gel strips, post-decolorization, are placed in an Eppendorf (EP) tube and washed once with a mixture of 50mM NH4HCO3 and ACN (1:1).
b) Complete dehydration is achieved by oscillating the strips in 100% acetonitrile, followed by the removal of excess liquid, and vacuum drying for 5 minutes.
c) A 150μL solution of 50mM DTT (dithiothreitol) is added, submerging the gel blocks. After vigorous shaking until the gel blocks swell transparently, the mixture is incubated at 56°C for 1 hour.
d) Upon cooling to room temperature, excess liquid is removed, and 150μL of 100mM IAA (iodoacetamide) solution is rapidly added. The gel blocks are submerged, and the reaction proceeds in darkness for 45 minutes.
e) Washing steps involve a mixture of 25mM NH4HCO3, 50mM NH4HCO3, and ACN (1:1), each applied once, with final dehydration in 100% ACN until the gel particles turn white. A final vacuum drying step is performed for 5 minutes.
f) Enzyme reaction solution is prepared by diluting the enzyme stock solution (0.1μg/μL) in 25mM NH4HCO3. The amount of enzyme solution added is optimized to submerge the swollen gel blocks. After a brief centrifugation, the mixture is incubated at 4°C for 30 minutes.
g) Once the solution is adequately absorbed by the gel blocks, excess enzyme solution is removed. Additional 25mM NH4HCO3 (20μL) is added, and overnight digestion is carried out at 37°C.
h) Enzyme solution is collected, and the gel block is subjected to ultrasonication in 100μL of 30% acetonitrile/0.1% TFA for 15 minutes. The solution is aspirated, and a subsequent ultrasonication step is performed in 100μL of 60% acetonitrile/0.1% TFA. The two solutions are combined, frozen, and lyophilized.
ZipTip C18 Peptide Desalting:
a) The dried peptide sample is reconstituted in 30 μl of 0.1% trifluoroacetic acid (TFA).
b) The ZipTip is equilibrated by rinsing with 50 μl of 60% acetonitrile (ACN)/0.1% TFA for 10 cycles.
c) A subsequent wash with 10 μl of 0.1% TFA is performed for 10 cycles to ensure thorough cleaning.
d) The sample is aspirated and expelled from the ZipTip for 20 cycles, discarding the liquid.
e) Another wash with 10 μl of 0.1% TFA is repeated for 5 cycles to remove any remaining contaminants.
f) Elution of peptides is achieved by washing the ZipTip with 10 μl of 60% ACN/0.1% TFA into a new Eppendorf (EP) tube, followed by vacuum drying.
g) The desalted peptides are now ready for analysis through mass spectrometry.
Database Retrieval:
For accurate protein identification, the acquired liquid chromatography-mass spectrometry (LC-MS) data is matched against protein databases, specific to the organism of sample origin. Commonly used software tools such as MaxQuant, Proteome Discoverer, and Peaks facilitate this matching process.
a) Database Download: Protein databases corresponding to the sample's organism are downloaded from UniProt.
b) Software Utilization: Utilizing MaxQuant, Proteome Discoverer, Peaks, or similar software, the LC-MS data is aligned with the information stored in the protein databases.
c) Identification Process: The matching process involves comparing the mass of primary and secondary fragment ions of peptides in the database, leading to the identification of corresponding proteins in the sample.
d) Handling Incomplete Databases: In cases where a comprehensive protein database for the sample's species is unavailable, databases of closely related organisms or those of model organisms within the same genus can be employed for protein identification analysis.
Reference
- Shevchenko, Andrej, et al. "In-gel digestion for mass spectrometric characterization of proteins and proteomes." Nature protocols 1.6 (2006): 2856-2860.
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