Mass Spec + molecular processing Flashcards

Question

Why use double charges?

Answer 1

Double charged peptide precursor to create single charged fragment ions These fragment with the same frequency at each bond so more likely to get a complete sequence Usually fragment at weak peptide bond Create B ions (from N) and Y ions (from C)

Answer 2

CID- collision induced (collide with neutral molecules) ETD- electron transfer (electron transfer breaks bonds) ECD- electron capture PQD- pulsed-Q dissociation (allows analysis of low m/z, ions held at high Q for kinetic energy) Tandem MS is used to select a specific precursor

Answer 3

Tend to fragment along backbone Lose neutral groups such as NH2 and H2O Type depends on factors- sequence, internal energy and how introduced, charge state Fragments need to carry at least one charge C terminal charge- X, y or z ion N terminal charge- a, b or c ion

Answer 4

Y ions- add H2O + H For B add H+ PTMs add extra weight

Answer 5

``` Assume lowest mass is y1 Lys or Arg and will be +19 Y2= Y1 + AA Proceed to right May be possible dipeptide peaks Unassigned will create a B series Highest mass is parent ion - Lys/Arg ```

Answer 6

MudPIT- 2D LC by ion exchange and reverse phase before MS/MS. Allows selection from complex mixtures ICAT- labels cysteine residues Peak each for diseased and healthy protein peaks MS Then MS/MS to peptide sequence to identify mutation iTRAQ- isotope encoded covalent tags to the termini. Samples from different conditions at pooled before LC. Can use to determine protein concentrations from reporter ions in different conditions 1D PAGE used with MALDITOF/MSMS or LC and MSMS 2D PAGE used with MALDI MS MS or ESI MSMS

Answer 7

Formation of 80S initiation complexes eIF3 is the largest initiation factor with 13 proteins One called eIF3i is essential in vivo but not in vitro- does this have a regulatory role? Used nanospray LC MSMS of tryptic digest using an ion trap FTICR mass spec to identify subunits and PTMS. 29 sites, mainly core subunits.fourier transform ion cyclotron resonance determines m/z by the cyclotron frequency in a magnetic field. Q-TOF analysed intact complex for stoichiometry and identification of peripheral subunits

Answer 8

Multidimensional protein identification technology Ion exchange and then reverse phase HPLC Separation from complex mixtures MS/MS

Answer 9

Isotope coded affinity tag Isotopic labelling method Reactive group to label side chain e.g. Iodoacetamide Cys Isotopically coded linker and tag for affinity isolation Allows two different samples to be combined into one mixture

Answer 10

Isobaric tags for relative and absolute quantitation Covalently bonded to the n or c termini Samples pooled and fractionated by LC Levels of protein in each condition Single peak allows concentration analysis

Answer 11

Energy metabolism Protein secretion Redox balance Growth/death control Lower probability of clone when productivity increases

Answer 12

Genome Transcriptome Proteome Metabolome

Answer 13

Proteome is the complete set of proteins Larger than genome due to splicing and PTMs Two levels of complexity not in the genome: the structure and the functional interactions between proteins

Answer 14

Design of new biological parts | Redesign of existing

Answer 15

Mathematical model of a system, so that modifications could be tested in silico Define what characteristics a relevant synthetic cell would have Choose a target, fermentation, and a metabolic pathway Creates an engineered base strain Further engineering for nutrients, transporters, chaperones etc.

Answer 16

Division Genome transfer Metabolism

Answer 17

``` Increased viable cell conc (IVC) Enhanced qP (cell specific production rate) ``` Summary- number of cells and how long they last plus how much protein they make

Answer 18

79 proteins in cell lines examined to see correlation between mAB Conc of chaperones, cell signals, cytoskeleton increased Highest producing line showed highest unfolded heavy chains So maybe this was an unfolded protein response? Unfolded protein marker not found in immunoblots Individual cells within parent population were better functionally equipped USED FOR DIRECTED EVOLUTION

Answer 19

Single mRNA -> single polypeptide -> protein Used to examine CHO cell lines Higher producers showed lower turnover of RNA and protein, meaning that more was available to make active protein Only around 10-20% of cells turned the protein into active protein Shows most cells are bystanders

Answer 20

HC and LC DNA -> polypeptides Folding intermediates- half antibody and LC dimer Assembled antibody and secretion

Answer 21

Intermediates can create rate limiting | Stuck as intermediates

Answer 22

Intracellular and supernatant species detected by western blot with antibody which can detect HL and LC Levels of half antibody in supernatant show by product due to slow redox recycling LC synthesis- more LC dimers HC synthesis- mAB increases, less unwanted SO HC IS A LIMITING STEP

Answer 23

Similar ratios of productive and unproductive export Suggests a common unselective export mechanism Ratio of exported species determined by the intracellular LCHC ratio Secretion doesn't limit production, secretory machinery not saturated beyond ER

Answer 24

Lots of LC produced per hour But lots of RNA of HC per hour Higher amounts of HC RNA suggest that LC is being folded more quickly than HC

Answer 25

Used to slow synthesis Cold shock response Promotes folding and decreases aggregation Less protein produced, so more time to fold

Answer 26

Hsp104 from yeast into Cho cells Increase in secretory and intracellular protein production So we need to target machinery that helps to assemble proteins

Mass Spec + molecular processing Flashcards

(50 cards)