L9 - Proteomics Flashcards

1
Q
A
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2
Q

What is the purpose of running a mixture of proteins on a gel in proteomics?

A

Proteins are separated by size so that the gel can be sliced into sections for subsequent analysis.

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3
Q

What role does in‐gel digestion play in protein sequencing?

A

In‐gel digestion uses trypsin to cleave proteins into peptides for mass spectrometric analysis.

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4
Q

Where does trypsin specifically cut proteins?

A

Trypsin cleaves on the C-terminal side of the basic amino acid residues lysine and arginine.

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5
Q

How are peptides separated after digestion?

A

The peptides are separated by chromatography before entering the mass spectrometer.

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6
Q

What does MS1 measure in a mass spectrometry experiment?

A

MS1 records the mass-to-charge ratio of the intact peptides.

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7
Q

What is measured during the MS2 phase of mass spectrometry?

A

MS2 measures the mass-to-charge ratios of fragmented peptide ions produced by smashing up the peptide.

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8
Q

How does the computer determine the identity of a peptide in MS/MS?

A

The software compares experimental spectra (MS1 and MS2) with theoretical spectra generated in silico from a provided protein list.

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9
Q

What is the ‘score’ or PEP in the context of MS/MS analysis?

A

It is a value that indicates how well a real spectrum fits a theoretical spectrum.

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10
Q

What is a decoy database and why is it used in proteomics?

A

A decoy database is a reversed version of the real protein list, used to estimate the rate of false-positive identifications.

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11
Q

Why must the protein search space be carefully defined in proteomics?

A

If all known proteins were searched, the decoy list would be excessively large, resulting in many false-positive hits.

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12
Q

What does a 1% False Discovery Rate (FDR) imply?

A

It implies that approximately one in every 100 reported matches is likely to be a false positive.

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13
Q

How is SILAC used to enhance immunoprecipitation experiments?

A

By differentially labelling samples, SILAC allows discrimination between specific and non-specific protein interactions.

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14
Q

What is the purpose of fusing a viral protein (e.g. NS1) to EGFP in these experiments?

A

The fusion protein is used as bait to pull down interacting proteins, which can then be quantitatively compared using SILAC.

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15
Q

How are non-specific interactions distinguished in SILAC-based immunoprecipitation?

A

Non-specific binding proteins typically show a 1:1 ratio between labelled samples, unlike specific interactors.

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16
Q

What is the benefit of combining proteomics and RNAseq data?

A

It allows the detection of anomalies where mRNA levels do not correlate with protein abundance, revealing post-transcriptional regulation.

17
Q

How can integrated omics data indicate active protein degradation?

A

A protein whose transcription remains constant while its protein level declines suggests targeted degradation.

18
Q

What example was provided to illustrate protein degradation without transcriptional change?

A

The apparent disappearance of MRE11 in adenovirus-infected cells.

19
Q

Why is integration of omics data important in understanding virus-host interactions?

A

It provides a comprehensive view that can uncover novel targets and mechanisms not evident from a single type of data.

20
Q

How can deep sequencing refine proteomics analysis in adenovirus research?

A

It enables re-inference of the proteome by identifying proteins that may be absent from the official protein list.

21
Q

What was discovered using combined proteomics and RNAseq approaches in adenovirus?

A

A novel adenovirus protein that had not been previously reported was detected.

22
Q

Why might an adenovirus protein be missing from the official list yet detected by these techniques?

A

The integrated approach can reveal proteins that standard databases overlook due to limitations in annotation.

23
Q

How did proteomics contribute to the understanding of SARS-CoV-2 strains?

A

Proteomics revealed the presence of two SARS-CoV-2 strains, including one with a deletion in the spike protein.

24
Q

What is the significance of the deleted region in the SARS-CoV-2 spike protein?

A

The deletion alters the peptides generated and has been linked to differences in viral pathogenesis.

25
Q

How was the existence of the deleted spike protein version confirmed?

A

By detecting the specific peptides corresponding to the deletion using MS/MS.

26
Q

What two new techniques are making a significant impact on modern biological research?

A

Next Generation Sequencing and high throughput tandem Mass Spectrometry.

27
Q

How do these techniques complement each other in integrated omics?

A

They allow simultaneous analysis of genetic and proteomic content, providing a detailed picture of cellular processes

28
Q

Why is integrated omics especially valuable in virology?

A

It enables researchers to monitor viral gene expression, protein changes, and host responses on a large scale.

29
Q

What is one major benefit of using quantitative proteomics in virus research?

A

It reveals alterations in protein abundance that might not be apparent from gene expression studies alone.