L9 - Proteomics Flashcards
What is the purpose of running a mixture of proteins on a gel in proteomics?
Proteins are separated by size so that the gel can be sliced into sections for subsequent analysis.
What role does in‐gel digestion play in protein sequencing?
In‐gel digestion uses trypsin to cleave proteins into peptides for mass spectrometric analysis.
Where does trypsin specifically cut proteins?
Trypsin cleaves on the C-terminal side of the basic amino acid residues lysine and arginine.
How are peptides separated after digestion?
The peptides are separated by chromatography before entering the mass spectrometer.
What does MS1 measure in a mass spectrometry experiment?
MS1 records the mass-to-charge ratio of the intact peptides.
What is measured during the MS2 phase of mass spectrometry?
MS2 measures the mass-to-charge ratios of fragmented peptide ions produced by smashing up the peptide.
How does the computer determine the identity of a peptide in MS/MS?
The software compares experimental spectra (MS1 and MS2) with theoretical spectra generated in silico from a provided protein list.
What is the ‘score’ or PEP in the context of MS/MS analysis?
It is a value that indicates how well a real spectrum fits a theoretical spectrum.
What is a decoy database and why is it used in proteomics?
A decoy database is a reversed version of the real protein list, used to estimate the rate of false-positive identifications.
Why must the protein search space be carefully defined in proteomics?
If all known proteins were searched, the decoy list would be excessively large, resulting in many false-positive hits.
What does a 1% False Discovery Rate (FDR) imply?
It implies that approximately one in every 100 reported matches is likely to be a false positive.
How is SILAC used to enhance immunoprecipitation experiments?
By differentially labelling samples, SILAC allows discrimination between specific and non-specific protein interactions.
What is the purpose of fusing a viral protein (e.g. NS1) to EGFP in these experiments?
The fusion protein is used as bait to pull down interacting proteins, which can then be quantitatively compared using SILAC.
How are non-specific interactions distinguished in SILAC-based immunoprecipitation?
Non-specific binding proteins typically show a 1:1 ratio between labelled samples, unlike specific interactors.
What is the benefit of combining proteomics and RNAseq data?
It allows the detection of anomalies where mRNA levels do not correlate with protein abundance, revealing post-transcriptional regulation.
How can integrated omics data indicate active protein degradation?
A protein whose transcription remains constant while its protein level declines suggests targeted degradation.
What example was provided to illustrate protein degradation without transcriptional change?
The apparent disappearance of MRE11 in adenovirus-infected cells.
Why is integration of omics data important in understanding virus-host interactions?
It provides a comprehensive view that can uncover novel targets and mechanisms not evident from a single type of data.
How can deep sequencing refine proteomics analysis in adenovirus research?
It enables re-inference of the proteome by identifying proteins that may be absent from the official protein list.
What was discovered using combined proteomics and RNAseq approaches in adenovirus?
A novel adenovirus protein that had not been previously reported was detected.
Why might an adenovirus protein be missing from the official list yet detected by these techniques?
The integrated approach can reveal proteins that standard databases overlook due to limitations in annotation.
How did proteomics contribute to the understanding of SARS-CoV-2 strains?
Proteomics revealed the presence of two SARS-CoV-2 strains, including one with a deletion in the spike protein.
What is the significance of the deleted region in the SARS-CoV-2 spike protein?
The deletion alters the peptides generated and has been linked to differences in viral pathogenesis.
How was the existence of the deleted spike protein version confirmed?
By detecting the specific peptides corresponding to the deletion using MS/MS.
