Proteomics Analysis Flashcards
What is proteomics
The study of the proteome, the complete set of proteins produced by a species, organism or cell type, using technologies of large scale protein separation and identification
What is expression proteomics
Identifying levels of proteins
Quantifying the proteome in 2 or more states (e.g. diseased and normal)
What is functional proteomics
Identifying protein-protein interactions
Quantifying these reactions to determine protein function
What makes up the proteome
All the different protein forms expressed at a given time in a tissue/cell or organism.
Subtypes - phosphorylation, methylation and other post-translational modifications etc
How are all the proteins identified
To identify all proteins at a given time you first need to extract them from the cells or tissue they’re in
Proteins may be in different cellular compartments -
Nuclei
Cytoplasm
Mitochondria
Why are detergents useful in proteomics
Use detergents to break open lipid bilayers of cells and physical means to disrupt connective tissue or extracellular matrices
What are the two types of detergents used
SDS
Triton-X
Both get inbetween phospholipid bilayer and break it apart.
How can the proteins be extracted from their compartment (nucleus/cytoplasm/ribosome)
Detergents
Physical disruption
Give two types of physical disruption
Homogenise
Sonicate
How does sonicate access proteins
Rod vibrates at high frequency and break cells apart
Very good at breaking open all membranes
How does homogenise work
Can break cell walls apart
Once the cells are fractionated, what is carried out
Density grade centrifugation to isolate the organelles
How are the proteins analysed
Using gel electrophoresis. Separates them based on mass.
What is SDS-PAGE
SDS unfolds and coats the protein in negative charge.
Proteins own charge is irrelevant
The mobility of where the protein lies in electrophoresis is dependent on the mass. Can calculate mass based on the known mass of the proteins around it.
The smaller they are the quicker the flow through the gel towards the positive electrode.
What is carried out after SDS-PAGE
Stain them with silver (more sensitive) or coomassie brilliant blue to see the gel bands (protein)
What is an issue with SDS-PAGE
Only separates on mass and if you have 1000s of proteins it would be hard to differentiate what proteins are present and its quantity.
What is 2D gel analysis
Can look at mass and charge or proteins and databases are used to identify the protein spots
What can 2D gel be used for
2D gel electrophoresis can be used to identify protein changes in response to a drug by calculating the entire liver proteome before treatment and the proteome changes afterwards using 2D gel
Can identify harmful changes that occur without physical changes to organs
How are proteins identified when no reference is available for the cell type or organism
Using one of -
HPLC
MALDI-TOF MS
LC-MS/MS
N-terminal amino acid sequencing
What is high pressured liquid chromatography (HPLC)
Allows you to very precisely separate proteins that are very similar (e.g. size, shape, hydrophobic etc)
HPLC tube at high pressure which a buffer flows through and separates the proteins by flowing through that buffer. Detected and appear on monitor protein A,B,C
Larger first to smaller.
Where has HPLC been used clinically
Guthrie test - sickle cell
HPLC allows variants of Hb to be identified and quantified
HPLC test can identify Hb F, A, S, C, E, and D from dry blood spots
Newborns with certain patterns of “at risk” Hb proteins will be analysed further
What is Matrix-Assisted Laser Desorption/Ionisation – Time Of Flight Mass Spectrometry
(MALDI-TOF-MS)
Sample added to matrix plate and hit with laser which vaporises it.
In a vacuum a current is put through which ionizes the vapourised sample and is accelerated due to its ionization and hit a detector.
The smaller molecules hit first - this can help identify the size
The charge of these proteins can be determined by where on the deflector the molecules hit.
What is LC-MS/MS
Combining liquid chromatography with tandem (2 rounds) mass spectrometry.
Liquid chromatography to separate proteins
Mass-spec to identify individual proteins
Why is LC-MS/MS useful
Gives information about the specific sequence of bases within that protein which can be used to identify unknown proteins based on sequence similarity
How does LC-MS/MS give specifics about protein sequence
The ionized fragments are passed through a collision cell to fragment them even more and are then analysed again using MS to obtain its sequenece.
What are protein microarrays
Similar to genomic microarrays however, much more difficult to produce.
Allows detection of many proteins with different properties in parallel using antibodies.
Can also use proteins that bind other proteins to find binding partners or protein complexes instead of antibodies.
Very variable and useful.
Where can proteomics be used in future
To test drugs to see which ones bind to the desired protein the best.
Why was RBCs investigated with ion trap mass spectrometry
Wider reading - kKHNIAVSHILL 2004
The RBC has been studied in depth regarding its spectrin membrane giving its elasticity and lack of organelles.
Now trying to discover proteome to help in the treatment of haem disorders.
What techniques were used to discover the erythrocyte/RBC proteome
Wider reading - kKHNIAVSHILL 2004
LC-MS/MS -
Combining liquid chromatography with tandem (2 rounds) mass spectrometry.
Liquid chromatography to separate proteins - using charge at one side of buffer to pull them through (largest first)
Mass-spec to identify individual proteins by collision to break them into peptides/ionized fragments and determine their AA make up by doing MS again.
These peptides were grouped with their proteins or origin and their abundance and molecular mass recorded and checked via proteomic database.
What challenges did LC/MS/MS discover about the RBC proteome
High abundance proteins like haemoglobin make the detection of smaller proteins difficult using LC/MS/MS. The smaller ones usually not picked up in high-pressured liquid chromatography even if within the correct range of sensitivty for the mass spectrometer.
The erythrocyte had to be broken down into cytoplasmic proteins and membrane skeleton proteins
What benefits did LC/MS/MS have over 2D electrophoresis when looking at the erythrocyte
2D electrophoresis cant discover glycosylated transmembrane proteins - theyre underrepresented in these gels. Can be found in LC/MS/MS though
Give some benefits of HPLC for sickle cell
WIDER READING - EASTMAN 1996
The Hb is eluted in water to seperate the haemoglobins, this is alsu used for PKU detection therefore not a lot of blood is required.
Isolates Hb on the collection card as other proteins cannot penetrate the resin on the card.
Variant haemoglobins can still be accurately identified from sickle cell even if they have similar detection times as pathological phenotypes on HPLC.
How are pharmacoproteomics being used to help discover new drugs
Using transcriptomes to see what proteins are translated from mRNAs however this is difficult as no protein applificaiton available like PCR.
What is one limit of 2D electrophoresis in pharmacoproteomics
far too many proteins at low expression levels
are overlooked. Typically, only 2000 or so of the most
abundant proteins in a particular cell or tissue can be
reliably separated and identified.
These seperated proteins are then identified using MALDI-TOF
Where has 2D electrophoresis helped clinically
WIDER READING - Anderson 1992
methapyrilene, an antihistamine and sleep aid withdrawn from the market after it was discovered to be a potent liver carcinogen in rodents, was shown by 2DE to target the hepatic mitochondrion by forming protein adducts specifically in this organelle. The
nature of the protein modification indicated that it was covalent and resulted from binding of a negatively charged adduct. This study raised the possibility that the
reactive metabolite, by attacking mitochondrial but not nuclear DNA, could exert its genotoxicity in an unconventional way. Detection of the protein modification by 2DE demonstrates the distinctiveness of this proteomic method
in detecting and quantifying drug-related processes that
result in the formation of reactive metabolites.
What did 2D electrophoresis show regarding ovarian cancer treatment
Wider reading - Hu 2002
Manymycin (farnesyl transferase inhibitor) response in ovarian cancer cells. The manymycin helps treat ras-mutated human tumours.
Using 2DE analysis of total proteins from
treated and untreated cell lines followed by MS identification of differentially expressed proteins, heat shock protein
70 (HSP70) was shown to increase following treatment.
Combined with data from other studies, this suggests that
HSP70 may enhance resistance and account for the altered
sensitivity of certain cancers to farnesyl transferase inhibitors.
What did 2DE help discover regarding statins?
WIDER READING - Steiner
HMG-CoA reductase inhibition caused an induction response elsewhere in the cholesterol synthesis pathways - cytosolic HMG-CoA synthase and isopentenyl-diphosphate delta-isomerase (new drug targets to lower cholestrol)
What is the yeast 2-hybrind method
WIDER READING
Used to analyse protei-protein, protein-DNA and protein ligand interactions.
A DNA binding domain and activation domain (usually in a transcription factor are cloned in separate vectors
The protein of interest usually cloned in the binding domain vector and the prey consists of all the members of a cDNA library that are cloned as fusion proteins in the activation domain vector. When they interact the binding and activation domains are brought close enough together to promote transcription and these yeast clones containing the transcribed proteins are selected
How was the yeast two-hybrid system used in breast cancer
WIDER READING
Tumor cell-derived membrane proteins were isolated, separated on 1D gels, digested with trypsin, and identified by MS. Nearly one-third of the 501 proteins identified were previously uncharacterized (eg, ESTs of unknown function). Three of these uncharacterized proteins were analyzed further for interacting protein partners using the yeast two-hybrid system. All three were shown to interact with known (ie, well-characterized) proteins, thus leading to a clearer understanding of their EST-encoded partners and their potential roles in cancer.
Why is proteomics more challenging than genomics?
WIDER READING
The genome is relatively constant while proteins differ from time to time and cell to cell.
Also, transcriptomics (mRNA) doesn’t always transcribe to proteins so not reliable.
Also posttranslational modifications.
How was MALDI/TOF used in necrotising enterocolitis
Wider reading - Dhillion 2007
Found in premature children of unknown cause presents like sepsis.
Used MALDI/TOF to identify NEC associated fecal microbiota to help with a quick diagnostic tool for the future
Findings - clostridium and klebisellia were the main causes along with an increased use of CPAP
How was MALDI/TOF used in pancreatic cancer identification?
Wider reading - zhong 2015
Identified a membrane protein - identified 55 proteins which associated with cellular differentation and apoptosis.
Prohibitin 1 (mitochondrial membrane protein) identified and correlated with pancreatic cancer - higher in cancer patients
Therefore may be able to be used as a biomarker for early cancer detection
How was MALDI/TOF used in antibiotic resistance
WIDER READING
Used for drug resistance checks in beta lactams (penicillin family) -
The MALDI/TOF detects the presence of carbapenemases, which indicates drug resistance to standard antibiotics. It is predicted that this could serve as a method for identifying a bacterium as drug resistant in as little as three hours. This technique could help physicians decide whether to prescribe more aggressive antibiotics initially.
How was proteomics used in rice
2D gel electrophoresis analysis was used to reveal new proteins in both leaf and stem tissues after jasmonic acid treatment (damages self-defense mechanism of rice).
Reductions in RuBisCo subunits in the stem tissues which was reversed by kinetin (free radical scavenger).
Shows taht jasmonate affects defense related gene expression in rice seeds
