Proteomics Flashcards
What is the proteome?
The entire protein complement of a cell line, tissue, or organism. A proteome undergoes continued surveillance and modification.
Why characterise the proteome when attempting to understand (and target) disease?
Proteome imbalance may result in human disease. Sensitive analysis of proteins can provide a window into disease pathogenesis, which is inaccessible by genomic methods.
How can we analyse proteomes?
Mass spectrometry.
Why mass spectrometry?
Specificity of identification, simple reproducible workflow, extreme sensitivity extending to the single species (an ion).
What are the steps to proteomics?
Protein mixture, digest peptides, peptide chromatography, MS/MS acquisition, data analysis.
What are the methods of sample preparation?
Proceed to separate peptides by liquid chromatography (HPLC) to simplify MS analysis.
How do we determine protein identity using mass spectrometry?
Very sensitive can detect the smallest changes. Trypsin cleaves arginine and lysine. Peptides are dip fed from HPLC to mass spectrometer. Mass analyses m/z ratio.
What is the soft ionisation approach, electrospray ionisation?
Charged droplets of protein solution are produced which shrink as the water evaporates. Freely hovering protein ions remain. Peptides are transferred from liquid to gas to remain intact.
How are ions detected?
Ions hit an electron multiplier, which is converted to an electric current and subsequently a mass spectrum.
How do we separate peptides of the same empirical formula?
Organised fragmentation. Cleave off one amino aid at a time. Phosphoric acid is lost.
How do we exploit tandem arrangments to arrive at peptide sequence information?
Ion source to mass analyser 1 to fragmentation. Most commonly done with collision induced dissociation. to mass analyser 2.
What is CID collision induced dissociation?
Accelerating ions and causing them to collide with neutral gas molecules such as he, n, O2. Leads to dissociation of ion into smaller fragments. Analysed for information on the original structure.
How many peptides are sequenced in a run?
10,000s. Date interpreted by bioinformatics.
How is MSMS used to probe post translational modifications?
For phosphorylation a neutral/loss scan. Phosphoric acid fragments more readily than peptide bonds. For ubiquitination. Missed tryptic cleavage at the specific lysine residue of the substrate when ubiquilytated.
What is the problem with analysis of PTMs?
Low abundance, poor ionisation characteristics. Low detection frequencies.
What are ways to improve analysis of PTM?
Metal oxide affinity enrichment of the phosphoproteome. Bind, wash, release, MS. OR immunopurification of ubiquitylated peptides using an antibody directed K-GG.
What are the variables of a label free sample?
Instrument time: more, wet lab complexity and time: little, comparability of samples: difficult, data analysis: complex, study design: flexible.
What are the variables of a labelled sample?
Instrument time: less, wet lab complexity and time: medium, comparability of samples: easy, data analysis: complex, study design: fixed.
How is metabolic labelling done?
Grow in heavy light medium, prepare protein lysates, combine samples, cleave with trypsin, peptide enrichment, LC-MS/MS, data analysis.
What is chemical labelling?
Label each sample separately. Peptide of interest will always elute and be present at the same time. Fragmentation Reporter cleaved away from peptide. Separate analysis of reporter. Allow comparison of all 4 proteins. Abundance of reporter directly proportional to abundance of sample.
How can we apply MS to the clinic?
As a tool to probe protein structure and function- more basic research. Broader omics analysis- lipids/ metabolites.
