Mass Spec + molecular processing Flashcards
Components of a mass spec
Ion source
Mass analyser to separate
Detector
Mass accuracy
Differs between machines
0.01% accuracy
For a 1000 Da peptide mass, will see +- 1Da
This +- is described as ppm
Fast atom bombardment
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
MALDI
Uses a 337nm UV laser Sample in cyano-Hydroxy cinnamic acid to charge Tolerant of low salt and detergent Pulsed beam Soft technique
ESI
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
Mass charge equation
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
MALDI-TOF
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
Creation of MALDI samples
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
Analysis of oligosaccharides by MALDI MS
Different forms of human interferon gamma
Used to observe different isoforms
different drug targeting uses
N-C terminal sequencing
Top down signal sequencing delivers N/C terminal sequence
Doesn’t need proteolytic digestion
Types of mass analysers
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
Quadrupoles
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
TOF equations
t= (m/2zV)^1/2 d m/z = 2Vt^2 / L^2
Quadrupole TOF analyser (Q-TOF)
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
Ion trap mass
Ion trapping devices that use a 3D quadrupole field to trap and mass analyse ions
Wolfgang Paul
Injected into or created in interior
Detectors
Used to use film
Now use ion channels and electron multipliers
When struck these produce a secondary electronic signal via an emission when struck
Protein ID by MS/MS
Peptide fragments are sequenced using a Mascot algorithm
Then queried against database
Types of search and data
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
Calculating peptide masses
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
Tandem mass spectrometry
Q-Q
MS (magnetic sector)-Q
Q-TOF
TOF-TOF
Resolution
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
Sequencing by MS methods
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
Ms/Ms for protein identification and PTMs
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
Why use trypsin for MS?
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
Why use double charges?
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)
Methods used for fragmenting peptides
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
What happens during peptide fragmentation
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
How to calculate +1 charged product ion mass
Y ions- add H2O + H
For B add H+
PTMs add extra weight
MSMS interpretation
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
Multidimensional LC and MS/MS
Gel pairings
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
Studies using MS
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
MudPit
Multidimensional protein identification technology
Ion exchange and then reverse phase HPLC
Separation from complex mixtures
MS/MS
ICAT
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
iTRAQ
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
What makes a high producing cell line?
Energy metabolism
Protein secretion
Redox balance
Growth/death control
Lower probability of clone when productivity increases
The omics sciences
Genome
Transcriptome
Proteome
Metabolome
Proteomics
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
What is synthetic biology
Design of new biological parts
Redesign of existing
Building a production cell
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.
3 mains things that need to be considered in synthetic cells
Division
Genome transfer
Metabolism
How could we improve recombinant yields?
2
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
Comparison of mAB cell lines
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
Luciferase as a model system
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
Diagram of antibody production
HC and LC DNA
-> polypeptides
Folding intermediates- half antibody and LC dimer
Assembled antibody and secretion
Limits on recombinant antibody production
Intermediates can create rate limiting
Stuck as intermediates
Model IgG4 experiment
Changing the LC and HC rates
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
Observing the flux through the cell lines for mAB
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
Looking at RNA levels of the igg4 model
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
Hypothermic conditions on protein synthesis
Used to slow synthesis
Cold shock response
Promotes folding and decreases aggregation
Less protein produced, so more time to fold
Introducing chaperones to increase folding
Hsp104 from yeast into Cho cells
Increase in secretory and intracellular protein production
So we need to target machinery that helps to assemble proteins
