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Proteins > Mass Spec + molecular processing > Flashcards

Mass Spec + molecular processing Flashcards

1
Q

Components of a mass spec

A

Ion source
Mass analyser to separate
Detector

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2
Q

Mass accuracy

A

Differs between machines
0.01% accuracy
For a 1000 Da peptide mass, will see +- 1Da
This +- is described as ppm

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3
Q

Fast atom bombardment

A 
Semi-hard
Liquid matrix
Caesium ions to desorb ions pass charge
Continuous ion beam
1000x less sensitive than MALDI
Can shear molecules giving fragmentation patterns
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4
Q

MALDI

A 
Uses a 337nm UV laser
Sample in cyano-Hydroxy cinnamic acid to charge
Tolerant of low salt and detergent
Pulsed beam
Soft technique
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5
Q

ESI

A

Protein, peptides, carbohydrates, small oligonucleotides
Intolerant of salt and detergents, charge interference
Coupled with LC or CE (capillary electrophoresis)
Picomolar to femtomolar sensitivity
Can be used with HPLC
Multiply charged species important for m/z
Multiple charging allows very large molecules in instruments with small mass range
Nanospray system 1ml/min
Either (M+H)+ or (M-H)- by adding Formic acid or ammonia
Amide and amino- positive detection
Acids and Hydroxyls that lose protons- negative detection

Nitrogen used as drying gas to concentrate charge
700-5000V

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6
Q

Mass charge equation

A 
m/z = (MW+NH+)/n
M/z is the mass to charge ratio
n is number of charges
MW of parent molecule
H+= 1.00785 Da
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7
Q

MALDI-TOF

A

Time between pulse and detection
Time is proportional to square root of m/z
0.005-0.001 accuracy
5000-20000 resolution
Sample mixed with UV absorbent
Believed to transfer ionised sample from condensed to gas phase, abalation from sample matrix

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8
Q

Creation of MALDI samples

A

Analate cocrystallised with molar excess of matrix compound
Irradiation by UV vaporises the matrix which carries the Analyte with it
In gas phase charged molecules directed to mass analyser
TOF separates m/z
Spectra with singly charged ion
Positive and negative modes
Tolerates salt and nonvolatile

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9
Q

Analysis of oligosaccharides by MALDI MS

A

Different forms of human interferon gamma
Used to observe different isoforms
different drug targeting uses

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10
Q

N-C terminal sequencing

A

Top down signal sequencing delivers N/C terminal sequence

Doesn’t need proteolytic digestion

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11
Q

Types of mass analysers

A

Magnetic sector analyser (MSA)- high resolution
Ion cyclotron resonance (FT-ICR)- highest resolution exact mass
Quadrupole analyser (Q)- can be followed by TOF for MS/MS. LOW RES.
TOF- no upper m/z limit, high throughout
Ion trap mass analyser (QSTAR)- good res, all in one mass analyser

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12
Q

Quadrupoles

A

Electro spray ionisation MS
Uses a quadrupole mass filter
One pair of negative rods, one pair of positive
Superimposed RF voltage 180 out of phase
RF/DC ration remains constant as voltages scanned
Only ions of certain m/z pass through the filter, others are thrown our of path
0-100 KDa
0.01 mass accuracy
500-2000 resolution

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13
Q

TOF equations

A 
t= (m/2zV)^1/2 d
m/z = 2Vt^2 / L^2
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14
Q

Quadrupole TOF analyser (Q-TOF)

A

Ion source -> skimmer -> hexapole
Quadrupole -> hexapole collision cell -> second hexapole
This allows selection of a particular ion
MCP detector and pushed towards reflectron
Reflectron reflects ions back to detector (electron multiplier)
Allows fragmentation of one peptide at a time

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15
Q

Ion trap mass

A

Ion trapping devices that use a 3D quadrupole field to trap and mass analyse ions
Wolfgang Paul
Injected into or created in interior

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16
Q

Detectors

A

Used to use film
Now use ion channels and electron multipliers
When struck these produce a secondary electronic signal via an emission when struck

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17
Q

Protein ID by MS/MS

A

Peptide fragments are sequenced using a Mascot algorithm

Then queried against database

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18
Q

Types of search and data

A

Peptide mass fingerprint- mass values from tryptic digest
Sequence query- one or more peptide mass values, partial Sequence, AA composition, MS/MS fragment ion masses
MS/MS ion search- raw MS from one or more peptides

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19
Q

Calculating peptide masses

A 
Sum of mono isotopic AAs
Add H2O because of N and C termini
Add H for charge
Add 16 for oxidised Met
Cys can be iodoacetylated because of reduced disulphides
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20
Q

Tandem mass spectrometry

A

Q-Q
MS (magnetic sector)-Q
Q-TOF
TOF-TOF

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21
Q

Resolution

A

Ability to see each ion as a peak
Larger molecules have low resolution
Separate and distinguish between ions of different mz values
Width of the peak is an indicator
Resolution = M/deltaM
Where deltaM is the width at half maximum

22
Q

Sequencing by MS methods

A

Fragmentation experiments can be carried out on
Tandem MSMS but also single analyser
Ion trap- fragmentation in ion trap before detection
MALDI with post source decay
TOF-TOF- with a collision cell

23
Q

Ms/Ms for protein identification and PTMs

A

Proteins isolated by gel or HPLC
Peptides ionised and sent to collision cell
Doubly charged ions selected
Ions fragmented through collision induced decay
Creates single charged daughter ions used to determine sequence

24
Q

Why use trypsin for MS?

A

Collision induced decay for peptides less than 2-3KDa is most reliable for MS-MS
Frequent trypsin sites
Putting basics residues at C terminus to fragment in a predictable manner
Also can distinguish between Leu and Lys

25
Q

Why use double charges?

A

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)

26
Q

Methods used for fragmenting peptides

A

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

27
Q

What happens during peptide fragmentation

A

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

28
Q

How to calculate +1 charged product ion mass

A

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

29
Q

MSMS interpretation

A 
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
30
Q

Multidimensional LC and MS/MS

Gel pairings

A

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

31
Q

Studies using MS

A

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

32
Q

MudPit

A

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

33
Q

ICAT

A

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

34
Q

iTRAQ

A

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

35
Q

What makes a high producing cell line?

A

Energy metabolism
Protein secretion
Redox balance
Growth/death control

Lower probability of clone when productivity increases

36
Q

The omics sciences

A

Genome
Transcriptome
Proteome
Metabolome

37
Q

Proteomics

A

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

38
Q

What is synthetic biology

A

Design of new biological parts

Redesign of existing

39
Q

Building a production cell

A

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.

40
Q

3 mains things that need to be considered in synthetic cells

A

Division
Genome transfer
Metabolism

41
Q

How could we improve recombinant yields?

2

A 
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

42
Q

Comparison of mAB cell lines

A

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

43
Q

Luciferase as a model system

A

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

44
Q

Diagram of antibody production

A

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

45
Q

Limits on recombinant antibody production

A

Intermediates can create rate limiting

Stuck as intermediates

46
Q

Model IgG4 experiment

Changing the LC and HC rates

A

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

47
Q

Observing the flux through the cell lines for mAB

A

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

48
Q

Looking at RNA levels of the igg4 model

A

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

49
Q

Hypothermic conditions on protein synthesis

A

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

50
Q

Introducing chaperones to increase folding

A

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

Proteins (8 decks)

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