Proteomics Flashcards
What is a proteome?
The PROFILING of the entire set of proteins within a system/cell at a given time and defined conditions. (discovery)
What are the different types of Mass Spectrometry acquisition modes?
The mass spectrometry (MS) acquisition modes are multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), data-dependent acquisition (DDA), and data-independent acquisition (DIA). MRM and PRM use user-defined precursor ions whilst DDA and DIA use MS-defined precursor ions. MRM follows single peptide selection, fragmentation, and single fragment selection for detection. PRM and DDA follow single peptide selection, fragmentation, and detection of all fragments. DIA follow multiple peptide selection, fragmentation, and detection of all fragments.
What is the shotgun (bottom-up) proteomic analysis method?
The shotgun proteomic analysis works from the bottom-up. It uses a detergent/cell lysis buffer to break the lipid bilayer and remove the large heme molecule in order to extract the protein content. The protein content is then estimated with the Bradford assay, Lowry method, bicinchoninic acid assay, or fluorescent-based assay. The protein extract is then fractionated with SDS-PAGE to purify and immunoprecipitate proteins, and then they are digested with trypsin to become peptide fragments. Followed by a clean-up step to remove trypsin. The peptide fragments are then lyophilised by freeze-thawing with liquid nitrogen at -80 degree celsius to preserve them to be chemically analysed by mass spectrometry in DDA/DIA mode which will collect their structural information and characterize complex peptide mixtures. Lastly, database matching will rebuild the proteins in silico and review the data’s quality and reproducibility.
Protein extraction, estimation, fractionation, digestion, clean-up, lyophilisation, chemical analysis, database matching.
What is liquid chromatography’s purpose in proteomics analysis?
To separate complex protein mixtures into a simpler ordered system. It uses an analytical LC proteomic column and solvent gradient profile.
What is the purpose of a mass analyzer/spectrometer (MS) in a proteomic analysis?
It detects, measures and quantifies peptides according to their mass-to-charge ratio, and records their spectral response as a function of total ion current (TIC) against retention time (RT).
What is the intact (top-down) proteomic analysis method?
Intact proteomic analysis is the top down method that uses matrix-associated laser desorption ionisation (MALDI) coupled with mass spectrometry (MS) on intact proteins. The intact proteins are mixed with the substrate material and then loaded onto the MALDI plate to be desorbed through pulse laser irradiation and ionised through protonation/deprotonation in the ablation gas. The ions are then accelerated to the MS to be detected, measured, and quantified (analysed) according to their mass-to-charge ratio and to record their spectral response as a function of the total ion current (TIC) against their retention time (RT).
What are the advantages and disadvantages of the top-down method?
The top down method allows for intact proteomic analysis, identification of protein isoforms, de novo sequencing, fewer false positives, complete protein coverage, and the characterisation of proteolytic processing events and post-translational modifications. However, the top down method is less sensitive with lower throughput, requires high sample purity, insoluble/big proteins are not applicable, expensive, produces complex data, requires sample preparation, and needs operator skill.
What are the advantages and disadvantages of the bottom-up method?
The bottom-up method can analyse complex protein mixtures, has higher throughput, collects more biological information, allows global coverage and characterisation of biological systems, can have MS2 level information, identification and database matching, and has greater analytical flexibility and control. However, the bottom-up method lacks chemical identification, has more false positives, proteins without tryptic peptides in the right mass window are missed, has less post-translational modifications and isoform information without the correct analytical strategy, requires sample preparation, and has a complex system, maintenance, calibrations, and system performance assessment.
A Bradford calibration curve with an equation of the line: 31.775x +0.3548. A protein solution was diluted 50-fold and 10µL was used for the Bradford assay giving an absorbance of 0.55617 at 575nm. What is the protein concentration of the original samples?
Equation: 0.55617 = 31.755x + 0.3548
Solve for x = 0.00063373
Therefore, x =0.0063373mg = 6.3373µg (1mg = 1000ug), the concentration per µL = 0.63373µg/µL (6.3373 divide 10µL)
Since a 50-fold dilution was performed, multiply 0.63373µg/µL by 50 to calculate the original protein concentration = 31.6866µg/µL.
What is Differential Gel Electrophoresis (DIGE)?
Differential gel electrophoresis (DIGE) is a 2-dimensional gel electrophoresis that labels up to 3 protein samples with different fluorescent dyes (Cy3, Cy5, Cy2) and then mixes them together. This is then loaded onto the isoelectric focussing (IEF) chromatography for the first dimension which separates the proteins into their isoelectric points. The gel strip is then transferred onto an SDS-PAGE which will separate proteins according to their size. Through multichannel imaging, the protein samples are detected according to their different fluorescence. Differential analysis is conducted through the binary shifts which may be horizontal (changes in isoelectric point), vertical (changes in size), or diagonal (changes in size and isoelectric point). Hence, DIGE allows the analysis of protein abundance, post-translational modifications, truncations, and any modifications that affect the protein’s size and isoelectric point.
What are post-translational modifications (PTM)?
PTM are chemical modifications that regulate protein activity. Such as phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, acetylation, lipidation, and proteolysis.
What factors are considered while designing a proteomic experiment?
Consider if the sample can be analyzed proteomically.
Consider if the sample replication and class structure are sufficient for analysis to answer the question posed.
Samples blanks should account for instrumental noise and background contamination.
Consider how many proteins are being detected.
Consider the method of analysis and if it can handle the number of data points.
How is quality control of trypsin digestion performed?
Bovine serum albumin (BSA) is added as it has a known response and is well-characterised, thus, if samples have a low protein abundance the BSA can help identify the source of error.
What proteomic method is preferred for large number of samples?
Label free quantification. Less sample preparation will prevent long processing and analysis time.
