Lecture 11: Proteomics Flashcards
What are the 2 main techniques used in proteomics?
- 2D- Polyacrylamide gel electrophoresis mass spectrometry
2. multidimensional LC-MS-MS
What is proteomics?
Proteomics is the study of the complete protein complement of a biological sample at a specific time. Proteomics provide a ‘snapshot’ of all proteins present and their abundances, not their expression.
What does proteomics not look at?
Proteomics does not look at the post-transcriptional modifications that can alter proteins such as methylation decoration, which can alter the proteins function.
What can proteomics be used for?
- Sensitive detection of proteins in the blood acting as biomarkers for disease.
- Cancer treatment, diagnostic and prognostic.
- Aid in the identification and initial treatment of diseases like dementia, Alzheimer’s and Parkinson’s.
- Used to identify and understand biochemical pathways.
What are protein made up of?
Proteins are polypeptide chains of amino acids held together by peptide bonds.
How many different amino acids are there?
20
what are the 2 types of amino acids?
- Essential AAs
2. Non-Essential AAs
What are essential amino acids?
Essential amino acids can not be formed by the body from the carbon skeleton of glucose, thus they must be obtained through the diet.
What are non-essential amino acids?
Non-essential amino acids can be formed by the body from the carbon skeleton of glucose, thus they are not necessary in the diet.
What makes amino acids difficult to characterise?
Their differences in physiochemical properties.
Why are intact proteins difficult to characterise?
Solubility issues (membrane proteins), activity (potease, phosphatase, etc.), aggregation (pH and ionic strength), and purity.
What is ‘Bottom-Up’ shotgun proteomics?
Process used to identify proteins in complex mixtures using a combination of high-performance liquid chromatography combined with mass spectrometry. Denature proteins so they are exposed to peptidases; trypsin cleaves protein at every tyrosine and arginine residue; identification of peptides in sample; peptides fed to a nano-HPLC MS machine for analysis and identification.
What are the benefits of qualitative proteomics by ‘shotgun’ techniques?
Aids in protein identification, identification of modifications made to the protein, and identifying any protein-protein interactions. Affinitive chromatography can be used to capture proteins and analyse qualitatively.
What are some of the common post-transcriptional modifications made to mRNA?
- Phosphor-proteomics
- Glycol-proteomics
- Redox-proteomics (Met, Cys)
- Methylation
- Acetylation
- Ubiquitination
What chemical modifications can be made to proteins?
Chemical modifications can include adducts from reactive intermediates.
What is the most abundant proteins in humans?
Hb
What is the 2nd most abundant proteins in humans?
Albumin
How do you remove unnecessary proteins from the blood plasma or analysis?
To remove unnecessary proteins from the plasma for analysis, specific enrichment methods based on chemistry must be used to remove larger proteins so as to allow analysis of particular, smaller, less abundant proteins. Multiple Affinity Removal System (MARS) removes the most abundant proteins in blood plasma to leave only the less abundant proteins.
Describe the NanoLC Workflow technique for the identification of proteins
- Denature, reduce and alkylate protein sample
- Trypsin digest to cleave protein to peptides
- Desalt/ SPE
- Analysis by nLC-MS/MS
- Database search against reference proteins
How is the peptide sequence of the unknown protein derived?
Peptide sequence is derived from predicted fragmentation from known database derived from genomic data.
What is 2D-SDS-PAGE?
2D- by sodium dodecyl sulphate polyacrylamide gel electrophoresis is a 2 step separation process of peptides by isoelectric point and then size
What are the genomic database search parameters?
- Taxonomy (human, mouse, e.coli, rat, etc.)
- Enzyme (typically trypsin, but others include Glu-c, Lys-C, etc.)
- Missed cleavages
- Fixed versus variable modifications (PTMs)
- Molecular weight (size) and pl (isoelectric point)
- Mass tolerance
How is MALDI-MS conducted?
Separation of peptides by isoelectric point and then size; cut out individual spots and analyse with MALDI-TOF-MS. Single protein digested with trypsin.
What are the limitations of MALDI-MS peptide mass fingerprinting?
- Automation required for 2D-PAGE
- Not quantitative; ionisation efficiency is dependent on peptide make-up eg. Peptides containing/ending with arginine ionise better.
- Works well for single proteins, which is rarely the case in a complex sample.
