Proteomics Analysis Flashcards

Question

How does LC-MS/MS give specifics about protein sequence

Answer 1

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.

Answer 2

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.

Answer 3

To test drugs to see which ones bind to the desired protein the best.

Answer 4

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.

Answer 5

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.

Answer 6

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

Answer 7

2D electrophoresis cant discover glycosylated transmembrane proteins - theyre underrepresented in these gels. Can be found in LC/MS/MS though

Answer 8

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.

Answer 9

Using transcriptomes to see what proteins are translated from mRNAs however this is difficult as no protein applificaiton available like PCR.

Answer 10

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

Answer 11

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.

Answer 12

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.

Answer 13

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)

Answer 14

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

Answer 15

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.

Answer 16

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.

Answer 17

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

Answer 18

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

Answer 19

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.

Answer 20

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