Mass spectrometry and proteomics Flashcards
What is a proteome?
The set of proteins expressed in a cell, tissue, organ, or organism at a particular time under specific conditions such as development, disease, or drug exposure
How does the proteome differ from the genome?
While the genome is largely static, the proteome is dynamic and changes based on conditions like cell type, developmental stage, and external stimuli
Why is the human proteome much larger than the human genome?
Due to alternative splicing of genes and post-translational modifications, which create multiple proteins from a single gene
What processes generate protein diversity?
Alternative splicing and post-translational modifications
Name some sub-disciplines within proteomics.
Protein expression profiling, interactomics, structural proteomics, functional proteomics, subproteome isolation
What is the main difference between traditional biochemistry and proteomics?
Traditional biochemistry studies single proteins; proteomics studies large numbers of proteins simultaneously
What is 2D-PAGE?
Two-dimensional polyacrylamide gel electrophoresis that separates proteins first by isoelectric point (pI) and then by molecular size
What are the steps in 2D-PAGE?
Separation by pI using isoelectric focusing (IEF)
IEF gel placed onto an SDS-PAGE gel
Separation by size using SDS-PAGE
What is 2D-DIGE and how does it improve on 2D-PAGE?
Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) labels different protein samples with different fluorophores, allowing them to be run together and directly compared on the same gel
What technology is used to visualise proteins in 2D-DIGE?
Fluorescence microscopy with different excitation wavelengths for different dyes
What are protein microarrays?
Solid surfaces (like glass slides) with microscopic spots containing different proteins used to study interactions like protein-protein, protein-lipid, and protein-DNA
Why is mass spectrometry used in proteomics?
To identify proteins by determining their mass and comparing it with database information
What is the basic workflow of mass spectrometry for proteins?
Ionisation → Mass analysis (based on m/z) → Detection → Data analysis
What are two types of protein microarrays and their uses?
Analytical arrays: detect protein expression and biomarkers (often using antibodies).
Functional arrays: study activities and interactions of purified proteins
What is m/z in mass spectrometry?
Mass-to-charge ratio
What happens to proteins during sample preparation for mass spectrometry?
Proteins are usually digested into peptides, often using trypsin
What is MALDI?
Matrix-Assisted Laser Desorption Ionisation, a ‘soft’ ionisation technique used for proteins and peptides
How does trypsin cleave proteins?
It cuts after lysine (K) or arginine (R) residues unless followed by proline (P)
What type of mass analyser is typically used with MALDI?
Time-of-Flight (TOF) analyser
How does TOF analysis work?
Ions are accelerated down a flight tube; lighter ions reach the detector earlier than heavier ions
What is peptide mass fingerprinting?
A method to identify proteins based on the unique pattern of masses of tryptic peptides compared to a database
What increases the chances of identifying the correct protein in mass fingerprinting?
Increasing mass accuracy and the number of peptides analysed
