MS 2 Flashcards
mechanisms of CID
Unimolecular dissocation or Direct stripping (direct stripping unfavorable)
FOM in collisional activation experiment
timescale, variance in energy, magnitude of energy, how energy distributed, efficiency, form of energy
How do you get velocity at center of mass
= momentum at center of mass and its the sum of the mass *momentum of product and neutral divided by the sum of their masses
What is true and useful at COM instead of lab
total E and KE are conserved and max E available is equal to relative KE (which is important because this is the energy that will turn into external or INTERNAL energy)
What is Q whats our equation for it and what does it mean
Q is change in kinetic energy - it indicates the amount of energy exchange between internal and external (going in or out) - it is N/(mp+N) * KE Lab SO as our N gets bigger this approaches one which means more efficient energy transfer (however larger ions can cause scattering)
What do different Q values relative to - mean
Q = 0 elastic collisoin, Q < 0 is inelastic - some energy into ion, Q>0 inelastic some energy out of ion
What is ngeative deflection determined by
Ke rel, v(r), and well depth - if low engouh can orbit (360 degree back scatter
What does interaction time determine
How likely excitation is - masseys adiabatic says the probability of energy transfer to a given mode is maximized
when the interaction time (tc
) ≈ that mode’s period of motion
tandem in space vs tandem in time
QQQ vs trap
In MS/MS what two broad things change fragmentation seen
Energy ++ activation method ( can also relate to time - can be slower or fast)
What are common types of MS/MS
CID/CAD: collision-induced dissociation, collisionally activated dissociation (gas)
SID: surface induced dissociation (surface)
IRMPD: infrared multiphoton dissociation (laser)
ABOVE IS VIBRATIONAL BELOW IS ELECTRONIC
UVPD: ultraviolet photodissociation (laser)
ECD: electron capture dissociation (electrons)
ETD: electron transfer dissociation (reactive anions
FOM in an MS/MS experiment
Efficiency, resolution, Ms^N how many transformations, fragments produced
What is a metastable ion
fragments outside source but before detection - if before mass analyzer detectable - if after r- will not be separated by mass
Benefits of using CID
can lose signal due to scattering and frag, but also REDUCES noise a LOT ,
Benefits of CAD
qualitative analysis, structural info, quant, ion chemistry, characteristic fragments for ID (eg small molecule, biopeptide , glycans etc)
Examples of MS/MS bioanalysis
peptide sequencing and PTMS, carbohdyrate structure. (cross ring cleavage)frag, lipied double bonds etc, nucleotides
How do nucleotdies fragment
B and W type ions means cut between phosphate backbone at at top of P and cleaving the 3’ OH
Where are protein x, y z (and a bc ions -
so a x is left of carbonyl, by is amide bond, and c z is N to c(side chain)
Mobile proton model
charge moves along protein backbone to cleave at most labile amide bond- charge directed
BEAM TYPE vs ION TRAP CID
beam type: (liek in QQQ or TOF - collision cell) more frags, less specific frags but more sequential, quick 10 us to 1 ms
IION TRAP _ lit, qit, frequency resonance applied - takes 10-100 ms, MORE SENSITIVE -(uses less energy)
How is Ion trap CID preformed
precursor isolation in time, dipolar RF signal applied at frequency of ions (for secular frequency to be activated) of interest with amplitudes to maximize products - (called a TICKLE ) –absorbs power form RF ripple
again lower collision energies
uses a bath gas
What are other types of CID besdies beam and ion trap
POST SOURCE DECAY in MALDI fragment in flight tube
Nozzle skimmer - high pressure at ion source - can undergo acceleration and frag
ICR methods- MECA _ multiple collision collisional excitation - a lot of low amp, on resonance pulses applied to precursor ions to increase internal energy for dissoc’
SORI - sustained off resonance irradiation - use an RF signal shifted to a lower OR one above and one below for multiply charged less translational energy so longer activation time, takes a long time - slow
VLECA - very low energy CA - multiple cycles of resonance excitation
WHAT determines what is seen in CID
energy and entroyp of fragmentation path, time window, internal energy dist
Process of Ion trap CID (from ion POV)
so again AC signal applied in resonance with secular frequency , increases its oscillation amplitude in trap so further from center - as such it feels stronger RF ripple which increases ion KF; then introduce bathg as - collides - transform KE to internal energy (increase ion temp) - upon doing this - no longer in resonance and collisionally called back down
DIPOLAR DC CID - what is it
displaced from center of trap but only axially
used because no resonance conditions, and first gen product ions continue to be heated which encourages consecutive dissociation
What is ion micromotion
the RF energy an ion feels when its displaced from the center which gives it increased kinetic energy
2 ways CA can occur
unimolecular dissoc and Direct stripping
FOM for CA
variability of energy, magnitude of enery, how energy distributed efficiency, time scale, form of energy
In veloctiy diagrams CA - what is Q and what does its relative values mean
Q is change in kinetic energy -
Q=0 is ELASTIC collision
Q<0 - inelastic collisions (some energy goes internally)
Q>0 super elastic collision (some energy is taken from internal energy)
With a larger ion in collision - what happens
ratio appraoches 1 - can get 100% of efficiency - Q max is N/m + N so this term approaches one largest Q max
2 benefits of using COM frame of reference
Change in direction is notable in scattering, change in KE is noicebale can tell if inelastic or super elastic
Why are scattering angles useful
They tell you about the collisional enery distribution
What is b and how does its values vary
B is impact parameter - indicating distance of closest approach of collision pair
LOW B means more interaction with repulsive part of potential
really low - causes direct back scattering
medium - causes forward scattering positive deflection
higher causes forward scattering (negative deflection) - dorrepsond to longer range attractive part of potential
How does V(r) and KErel effect deflection
lowers negative deflection and if collision low enough energy - they can stick and give a scattering angle of 360 degree
WHAT IS MASSEYS ADIABATIC CRITERION
Probabiltiy of energy transfer to a given mode is maximized when interaction time matches the modes period of motion
tc/T (interactiontime divided by period of motion)
so if Massey > 1 that means period of motion is larger, collision is slow takes too long relative to motion transition unlikely
Massey < 1 collision is too short for the internal motion
Massey = 1 perfect -
What does interactoin time determine -
type of excitation and mechanism
Which type of excitation is more likely and why (radiationless between electronic states or DIRECT of vibrational modes
Vmax range for electronci (the relative velocity for max probability) is 10kEV to 1000 KeV
vmax range for direct is 100 eV o 100 kEV - MUCH more attainable
TYPES of CIDS and notable differences
HIGH energy CID -fast activation 2-10 kEV - us time scale
LOW ENERGY - 1-200 eV, .5-1 ms
Trapping CID - 1-20 eV takes 10-100 ms
How does slow heating work from a diagram perspecitve
1) initial activation
- this increases the probability of activating events so it outweighs deactivating events (increases T internal)
- this continues and eventually reaches a stead state distribution of internal energies
IF some of the energy is above E0 - fragmentation will be observed
What are the steps(rate reaction wise) in slow heating frag and what determines the probability distribution
There is ACTIATIOn and then dissociation
if dissociation is rate limiting step THEN - we see a normal distribution - the amount of activate ions and pre activated reaches an equilibrium - there’s a lot of them to go to the next step
BUT if ACTIVATION is rate limiting - then we see a TRUNCATED boltzman distribution not a lot activated compared to how many will dissoc
Why does mechanism of activation matter
energy distributed differently - doesnt always behave statistically
can be (T-> E,, T->V or T to V and E (vibration or electric)
What are some aspects of T-V mechanism
COMPLEX formation - most efficent
fraction of KE that goes into precursor depends on DOF, complex lifetime
favored with low Kerel and is high efficiency Q/Kerel
LARGE B negative deflection
is like a lot of collisions with favorable massey parameters
*THIS essentially is our precursor and our natural collide and become a complex - while complexed or orbiting undergo a lot of favorable Massey collisions - but ultaimeyl they need to separate and when they do - how the energy is distributed matters and causes frag
What is possible vs what is probable
I think what is possible is more in terms of conservation of momentum mass etc - that stuff repulsion collsioisn B, KE rel WHAT Is probable relates more to timing (Tc)
Describe T->V Impulsive collision
These are called impuslive or binary and they are ELASTIC
positive deflection - SMALL B
lower efficiency than complex (it’s just one collision - KE REL a lot higher here can be
So here don’t need good massey parameters (so collision time can be shorter than period of vibration (faster) - BECAUSE HERE ITS INDEPENDANT - occurs indirectly though an elastic collision and the RECOIL energy is distributed into vibration
Describe t->V direct induction
LARGE B - little scattering - small energy transfer, very timing sensitive - not common ( I guess just flings by and somehow transfers vibrational energy in passing
Describe T-> E VERTICAL excitation
LARGE B again - little scattering
need interaction time < 10-14 s? (seconds?
slow enough to excite but not breakdown (these are not favorable for Massey - its too high to reach these as stated earlier) but still want high E
T->E Curve crossing - dsecribe it
- Collision must be slow enough to
allow transition to the upper,
excited state
can occur with low b
– But fast enough to prevent
crossing back down to the original
state
MOST likely for t-> E
probably at lower velocity - smaller b
can happen with tc < than period of vibration so large KeRel
List all of the mechanisms of energy transfer in CAD
T-> E is curve crossing, or vertical excitation
T-> V is complex formation, binary or impulsive and Direct
describe T->E + V curve cross + impulsive collision
Curve cross to excited state with an impulsive collisions (curve crossing on repulsive part of potential
- Energy partitioning can occur several ways
– Ion can be both vibrationally and electronically excited
– Ion can be vibrationally excited while target is electronically excited
Why should we analyze lipids
end product to upstream activities and as a such closer to the actual phenotype than something upstream
Also very ubiquitous /prevalent
also hallmark role is in membranes , inflammation, energy storage
What are some challenges in analyzing lipids
COmplexity (there’s a lot of them),
small changes such as just the difference of double bond position (isobars as well),
isobars,isomers
naming can be difficult,
quite a lipidome diversity depending on what you are looking for (eg racial);
Dynamic range can be difficult then [] range in a lipid profile
Stability - can derivitize or add anti oxidants
What are layers of lipid identification specificty
Class, sum composition (eg PE 36:1), FA identification, FA position, double bond position, stereochemistyr
Common lipid extraction
Folch vs the Bligh and Dwyer
They’re both just version of LLE - chloroform metahnol and water(avoid plastics!) can do multiple extracts
How are lipids quanted
not ISTD for all lipids so use one for many or do semi quant 1 pt cal curve or do RELATIVE quant
Typical setup for lipid anaysis
DI (SHOTgun) or LC (reverse phase or HILIC) - into ESI MS (HRMS orbitrap) or tandem MS
Pros of shotgun (DI analysis) vs LC
all area analyzed under the same MS conditions instead of across an LC run;
VERY QUICK
can do one IS per class
negatives of shotgun - isotope overlap, lipid aggergation
CHROMATOGRAPHY -
seperation great
quant can be more difficult need more IS per class
What is iterative exclusion MS for lipids
Do a full scan - take the 10 most abundant and REMOVE THEM - don’t analyze them then do another inejct - and then take the 10 NEXT most abundant - then exlcude those so on and so forth
What essential types of reaction does RRKM theory
2 reactions: Activation (bimolecular) and then reaction/fragmentation (unimolecular)
What is the Lindemann theory
We have our 2 reactions bimolecular actiation and then unimolecular fragmentation and the rate is PRESSURE DEPENDANT - at high pressure k1>k2 so its first order;
At low pressure k2>k1 so it’s second order overall
NOTE rate law is determined change in concentration of activated A* over time
What’s wrong with Lindemann thoery?
rates fall off at higher pressure ;
doesn’t include DOF (molecules already has some energy )
doesn’t account for energy level
doesn’t account for any specificity in which bond it takes place in
What did Hinshelwood add to Lindemann theory
Added internal energy - accounted for rotation and vibrational internal energy) - modeled them as oscillators - and modeled the probability of any given reactants activation by boltzmann distribution (does it have energy between E and E + dE)
So the above is just for one DOF BUT he also accounted for many DOF as well across all DOF at every collision
Strong collision approximation - what is it and where used
assuming energy of molecules completely randomized ti biltzmann- the activation rate proportional to boltzmann constant
Hinshelwood Shortcomings
Doesn’t account for effectives ness range of oscillators (1/2 are effective) -
Only dealt with K1 process - energy of activation
K2 has own issues not addressed assumed to be uniform when it’s not
RRK theory what did it bring to the table
Needed to allow for flow of energy so now molecule viewed as a system of loosely coupled oscillators (so here we get degeneracy - how many ways can we put J energy into S degrees of freedom) - how the energy distributed
Split k2 into 2 steps
1) Energized
2) activated
Difference between activated and energized molecules (K2 vs Kdouble dagger)
A double dagger is passing into final states (products) - A* (or K2) has sufficient energy to become activated without additional energy but needs to undergo vibrations before becoming activated
Problem with RRK theory
coupled oscillators is not clearly identifiable with normal modes, doesn’t incorporate magnitude of all frequency factors (higher ones)
RRKM theory what does it add
Adds transition state theory - on an energy surface once pass transition state - will continue in forward direction - wont recross in reversal
individual vibrational frequencies are considered explicitly
rrkm ASSUMPTIONS
Time for dissoc is long comparaed to everything else (formation, activation, redistribution ,
ASSUMES ergodic redistribution (so doesn’t work for ECD or ETD that doesn’t distribute across whole molecule)
Is ADIABATIC - takes place on single energy surface - motion is classical
there is a transition state point of no return
How to get reaction path degeneracy
number of bonds essentially - number of areas energy could be in
What is tightness of transition state and how does it affect reaction rate
essentially things that are more labile -e easier to cleave - as internal energy increases, looser transition sates go up more quickly than tighter - so rate goes up quicker
How does DOF affect rate
DOF decreases rate - more modes to spread energy across
How does critical energy effect reaction rates
higher critical energy are slower rates - typically not observed
How does rearrangemnt vs simple cleavage effect rate
Rearrangements are low critical energy so at low internal energy they are favored but at higher internal energy - simple cleavages are competitive/favored
Types of Energy shift (RRKM )
Kinetic shift - more energy than critical for frag is required to observe pathway (basically needs faster reaction rate to observe)
Thermal - less energy than for frag is required to observe (molecule already has own internal energy contributing)
Competitive - more energy than for frag needed to observe because there is a competing path way - want to drive desired pathway
Kinetic sotope effect
energy level (0 point) is mass dependant so critical energy for lighter masses takes less energy so rates for them is faster
calculate Mass Spec steps from QET
1)ID frag pathways and kinetic sceheme
2) rate constants and generate k vs E curves
3integrate k vs E curve over determined reaction time
3.5 generate breakdown curve - (ion concentration over range of energies)
4) determine potential energy distribution of formed ions and convoluted with breakdown curve to generate calculated spectra
MS - why use seperation
Orthogonality - MS can’t sep but can sep on column like isomers, chiral compounds
Efficient ionization (instead of ionizing all at once- separate over time - less matrix effects, decrease LOD
GC considerations for MS
needs to be volatilized, LARGE flow rate for MS need a splitter, column bleeding etc
LC van deemter considerations
eddy diffusion, longitudinal, mass transfer
Eddy diffusion small well packed columns,
longitudinal smaller flow path and higher flow rate
mass transfer - small particle size, low flow rate and heat
Issues with multi dimensional LC
run time
Compare MSI to other imaging modalities
So less resolution than a microscope, also generally not in vivo like x-ray, MRI etc
BUT the most specific out of all of them (so microscope level resolution with specificity (so ultimately good complementary technique)
MSI applications
molecular histology, ultra high spatial resolution, full body imaging
MSI work flow
cryosection, 10-20 um, - onto a slide, apply MALDI matrix - data acquisition
What is Histology directed acquisition (or profiling)
Tissue annotated by pathoogist before MS and then MS data only acquired from spots of interest (increase throughput) -
IMS FOM
mass resolving power, accuracy, sensitivity , dynamic range ,
SPATIAL REOSLUTION
THROUGHPUT
FILE DATA SIZE and storage
Goals in MSI sample prep (4)
analyte delocalization, tissue delocalize, physical deformity, optimize sensitivity + resolution
Types of Matrix application and considerations
Don’t want too wash - can cause extraction/delocalization;
Spotting - can be manual or robotic - Large crystals but good extraction
Manual spraying - not consistent
robotic sprayer - not the highest resolution, expensive but regular
Sublimation - smallest crystal size - good sensitivity
Matrix considerations for MSI
extraction while limiting delocalization
-analyte, crystal size
recrystallizing time
What sorts of things can we do with Ion IOn or ION Molecule MS/MS
charge permutation, induce dissociation (LIKE ECD) ORR perform synthetic chemistry
What is a charge permutation reaction
Basically taking or donating an electron or proton - effectively changing the charge of our analyte
Explain EIEIO and EED
So EIEIO - electron impact excitatin of ions from organics - basically intermediate energy interaction with an electron causes fragmenting (5-10ev)
EED is a higher energy e- essentially - EI ionization - causing our species to become + charged which can then capture an electron and fragment
ECD what is it
capture of a low energy electron(1-3 eV) - ; need to us a multiply charged species
for a peptide - this capture is 6 ev NON ERGODIC PROCESS - no redist
How can ou trap electrons for ECD
magnetic + weak electrostatic field in ICR allow for this (cant do in ion trap
HOW to ECD with no ICR
chimera cell - has a mag field area to trap electrons
Describe ECD mechanism
So 2 steps - e- arrives
depends on local intramolecular electrostatic potentials - to stabilize it (if not there e- cant attach) - coulombic and dipoles at work here-THIS WILL TYPICALLY BE like an n terminal or aan amine - AND THIS can h bond to ca rbonyl which makes this more likely (also directs next step)
2) - ONCE attached - Transfer time - so the positive attachment site is the RYDBERG orbital antenna in which the e- an go to acceptable orbitals to then cause cleavage (COMMON ONES are the C of a carbonyl (can have dipole there) -so the amide pi orbital* or S-S sigma bonds
- this second attachment Must be within radial shell of rydberg orbital and have electron BE that can overcome rydberg
3) this causes cleaveage because electornsi in this antibonding orbtial is repulsive for sigma *
For the amide pi - it lower the barrier to hemolytic cleavage (this will usually form an enolimine or amide
Cornell vs UW mech
so in ECD when electorn relaxing- UW mech it can go to long distance orbitals - Cornell mech says if H bonded to carbonyl then it’s going to go there
CID vs ECD
ECD more pTMs and better sequence coverage
c and z ons instead
ALSO on folded intact proteins - if stable can cleave one bond and still have it stay in tact
EC NO D (ALSO REDUCES CHARGE STATE
How does the mass calculation work for ECD (in terms of charge states)
So its the same M+ H / BUT keep in mind we’ve decreased the charge so for example could get a 5+ charge state but with 8 protons on it
Why might we use slow heating before ECD
as mentioned can get EC no D on intact folded protein - SO if use slow heating like CID, SORI or IRMPD can unfold it - thermal heating and THEN ECD (called HAWT ECD)
How does negative ion ECD work
Need a protein to be VERY lARGE - so negative charge repulsion is localized elsewhere I guess
What is EDD
EID (or EI like) but with anions - so makes it even more negative -low efficiency - minimal internal fragment mostly sidechain losses
UVPD
Fluoresence - back down to groundstate - amount of frag depends on fluorescence quantum yield
BREAKS C-I bonds so experiments might introduce an I which creates radical can CID for extra frag
Notable IRMPD stats
Can keep fragmenting and get consecutive frags - no collisional cooling (less major ions more losses of H2o , NH3 etc
What are characteristics of I/M reactions
HIGHLY exothermic
very high rates - typically between oppositely charged (obviously one of them needs to be multiply charged
ETD - how does it work
Cation reacts with an electron carrier -
electron carrier inrdouced (ETD REAGENT) via electrospray or APCI
ETD what do you see thats unique
retains S-S disulfides
AI ETID - what is it (actiated ion)(
its liek hot ECD but ETD
Proton Transfer
***In this reaction reagent is an anion and your analyte is donating a proton to it
Basically if have even electorn anion - can pull in a proton and can condense signal ION CONCENTATION (can control with applying a waveform and controlling velocity (decreasing so get it reduced to a certain number of states - you apply the waveform for the charge state you want)
can also charge invert
REACTION THERMODYNAMICS EQUATIONS
kc -= v * pi * (Z *Z *e^2 / 4 *pi *e *u ( v^2))^2
SO key parameters I guess are charge, velocity etc - SO z , mass velcotiy SO - we can control reaction rate with velocity
With Chemical derivitization in gas phase what is key about the reagents
The reagents mainly target primary amine funcationilities -
multi functional want them to be electrostatically sticky like sulfonate - reactive groups
example stick together then covalent complex AND THIS CAN ALTER FRAGMENTATION (eg effects mobile proton model because ergodic
other reactions can do in gas phase
protein synthesis, Hydorgen detuterium exchange, protonation
Basics of ION MOBILITY
seperate molecules based on how they can move against a buffer gas when driven by electric field (more compact faster)
Why do we use mobility for IM
Temperature and pressure variations - not very reproducible so use mobility as a comparable attribute and REDUCED mobility accounts for temp and pressure
CCS explain it
can relate drift time to CCS awhich is an area based measure (we convert drift time to CCS - which is in the units of angstrom Squared using the MASON SCHAMP EQUATION (in which there are a lot of constants and really the only change is drift time
What are the 3 types of Ion mobility
DRIFT TUBE, TRAVELING WAVE AND TRAPPED
EXplain drift tube
uses quadrupoles - and a mix of RF and DC to guide ions down tube (DC pushes down , RF to keep confined - constant electric field to keep flight time constant
SO initially have hourglass funnel - store ions have electrostatic gate to PULSE it
How can IMS be used for proteins
different charge state have different CCS (*the more unfolded they are)) -de to coloumbic repulsion so can see this and separate them
IMS/IMS - what is it
IMS - focus and CID? and IMS again
Pros and cons of DT IMS
pro - classic, highly accurate and reproducible
con: home built
variations
helium gas buffer expensive
EXPLAIN TWIMS
instead of continuous DC - use DC waves - low to high voltage to push
this means ions either go with a wave to the end or go some of the way and need another wave
pros and cons of TWIMS
allows it to be more compact, with same res
commercialized
easy to use
CON: no uniform electric field so cant get true ccs (need to calibrate to get it)
no tandem IMS
TIMS expalin it
Use an electric field to trap ions and use buffer gas to separate them (so actually bigger ions elute first
Pros and cons of TIMS
CAN get direct CCS values
commercially available
compact
Great resolving power
CONS: more complex
Pros and cons on using MS for protein protein interactions
Pros: speed, sensitivity, specificity, can do membrane proteins
cons: MW limit, gas phase vs solution (is structure the same>?
What is one way they confirm the gas phase vs in solution is similar
CCS values - compare CCS
Whats the solution to gas phase steps
Folded or unfolded structure - to low charge state to unfolded intermediate - to potentially refolded gas phase structure (or could be high charge state unfolded gas pase
ESI considerations in intact protein analysis
Voltage - lower voltage wanted to not unfold - heat less, don’t heat the capillary etc
for this nano ESI is preferred
ALSO - because smaller droplets so less fission events -less adducts etc (NORMAL ESI has a lot fo aggergates shaggy)
also normal ESUI + energy, will lose some structure
Whats non specific aggregations
PART OF THE CRM METHOD OF IONIZATION FOR NATIVE MSwhen concentration too hgigh makes aggregates - important because we want to see whats in vivo not stuff made in the MS (ideally concentration is one molecule per droplet
native ESI vs regular esi diffrences
Solution will be largely aqeous , a lot lower flow rate, 1-10 uM
When we do CID on our native protein what can happen
break non covalent linkages first - so might have a lot of non covaletnyl bound subunits - - so causes unfolding - surface area increased BUT these cause a MAJOR loss in mass and a major loss in charge as well - losing these usbunits
What do we need tofr the analysis of transmembrane proteins and how do we interface with MS analysis
They need detergents/lipids - we try to remove them during desolvation so no lipid adducts need CID right collisional energy (too high causes dissociation, and not using it causes unresolved charge states
What is another strategy that can be used for nativ eMS
ion mobility
CID vs SID
SID adds ihgher energy quicklly I guess but more importantly GET different subunit break down (eg a hexamer via CID can turn into monomer and pentamer whereas a SID can make trimers (also get other “mer” types)
Native mass spec interpretaiotn
need to be careful with things that look like like maybe distributions might just be phosphorylation for example but at multi charge state - so we’ll get a distribution the smallest will be isotopes which will be less than aunit - larger ones will be phosphorylation OR oxidation events small
What are some biophsycial MS tools
H/D exchange, chemial crosslink, covalent labeling, ECD and UVPD, also I guess Ion mobility, proton transfer etc
What is chemical crosslinking
Have two proteins - cross link them in vitro or in vivo - and then analyze th linked peptides - the IDEA is to map proximity of amino acids in natural state (eg see WHERE cross links can form can tell us the distance
How do you make protein refold in gas phase?
Do proton transfer - to bring charge state down - as charge state comes down - REFOLDS
What are some typical prtoeomics methods
SDS PAGE, Edman Degradation sequencing - not common
Western Blot - for presumptive ID
Describe western blotting
run SDS AGE - transfer the proteins to nitrocellulose membrane - incubate with antibody - (should bind to protein) - then incubate with 2ndary antibody (antibody for first antibody and should have fluorescent tag
now visualizable
Why is peptide mass finger printing by MALDI not accepted for Protein ID
cant distinguish peptides of same MW, PTM assignment difficult, sequence coverage etc, cant do mixes
Data dependant vs Data independant for proteomics
DDA is you see a specificpeak - you fragment it
Data indpenedant is for each MS scan you do an MS 2 scan of EVERYTHING but in smaller mass range windows for the second product scan (eg 25 m/z -) - SO LOTS OF DAT
Database search for proteomics
MASCOT and SEQUEST
How does database sarching work and what are important factors
protein sequenes are digested and fargmented in silicon making large peak list -
critical that enzyme work properly - over digest or missed cut sites can mess it up
need proper mass accuracy according to instrument
need PTMs or modifications
QUantitative methods for proteomics
DIGE, SILAC< ICAT< iTRAQ and label free
Explain SILAC
stable isotope labeling with amino acid in cell culture - basically have two cell cultures - one you only put in heavy labeled say c13 - so all proteins are made with that - you can then mix and then analyze the relative amounts of control vs the isotope labeled protein
there is aPUSLED and DYNAMIC form
pros and cons of SILAC
10 ug of protein, BEST quant, one experiment a lot of data, can do in vivo
CONSL need LC and high resolution instrumentation, lots of data, expensive
iTRAQ explain
isolate protein - digest - and label with a MASS TAG - labeling efficiency is really key
when do analysis - mMS/MS causes tag cleavage and then just the relative amounts of tags can be compared
pros and cons of ITRAQ
lots of info from one expeirment
cons: LABEL EFFICIENCY
Explain label free quant - pros and cons
no label - jsut relative concentration amounts - can do SPECTRAL COUNTING or area under the peak
Other more specific types of proteotmics anaysis besides quant and sequence
Modification and Linkage analysis
Resolution definitions
M / Delta M ; delta M being difference between two peaks of abundance with overlap defined (geg 50%)
RESOLVING POWER _ just peak width at x height (FWHM)
Mass accuracy
Mexp - M calc / M calc * 1 million
Expplain mass defect
From nucleur binding energy - ( e = mc^2)
note some have small positive defect (light elements) - most have negative
What is an isobar
same NOMINAL mass
What is an isotopologue
an isotopologue is an isotope that could have two variants eg 2 c13s or 1 Cl37 - and with high enough resolution we can distinguish due to mass defect
what is isotopic fine structure and example
the unique MS signature arising from naturaly occurring isotopes
SO for the A +1 would typically think C 13
but actually a mix of C13, N15, S33, H2 etc and each of those can be seen with their mass defect
What are other things that can cause MS distributions besides charge state and isotope
loss of a ligand liek phosphorylation , or glucose addition or loss
OR oxidation etc - especially consider these at higher charge states and mass they would add
so consider mass defect and how much larger it should be due to charge
Hohw can one clean up a difficult distribution they are trying to deconvlolute?
IION ION REACTIONS do charge reduction
How does one go from an M dot + to an even electron ion
So one atom is missing an electron so its + charged. So that electron and one electron from the cleaved bond go towards making a bond somewhere else SO
if you think of an alcohol the alpha carbon has a bond to the beta carbon - that bond one electron will donate to the other side of the carbon and the one electron on O will also help to make a double bond
So we’ve LOSt an electron in terms of oddness evenss (because the other one of that pair from alpha to beta goes to the cleavage product )
2 major types of cleavage pathways in EI
SIMPLE: cleavage of a sigma, induced or alpha bond
REARRANGEMENT: - radical or charge site
RESONANCE STRUCTURE RULES
atoms never move, can;t change overall charge
only electron move from lone pair or pi bonds
framework intact
WHERE IS THE INTIIAL ELECTRON REMOVED FROM
lowest ionization potential
so LONE PAIR> pi > sigma
What are the cleavages an OE can undergo (odd electron) (based on Positive charge and where lone electron is)
INDUCED vs ALPHA
INduced - attraction of electron pair that causes a heteroytic cleavage - MIGRATION OF CHARGE SITE - BASED ON + site)
Alpha cleavage - donation of a single electron to form a new bond causes HOMOLYTIC CLEAVAGE - migration of the unpaired electron site
What fragments results from ONLY induced or alpha cleavages
Even electron and neutral radical - would require rearrangement or multi bond cleavages for anything else
What happens in the case that an electron is removed from a sigma bond (direct dissociatoin
In this case the c-c bond weakens and fragments
One frag gets the remaining electron (neutral radical) and the other
Who gets the electron or the positive charge - how determined?
STEVENSONS RULES
Formation of stable ion
formation of stable neutral
size of natural loss
1-2 are thermodynamic
3 is kinetic
SO the charge will be retained on that with the lowest ionization potential so dependant on heat of formation for our ionic products (so a tertiary carbon is going to be the best (1>2>3)
The energies of our Ion are going to vary A LOT MORE THAN our neutral so that will most likely dictate it (even though same rules apply for neutral)
Dsecribe an induced cleavage:
Means our + dot is INDUCING the electorns of a nearby bond to stabilize it -
SO - those electrons go there and stabilize that making a NEUTRAL RADICAL - and the electrons leave the other portion making a cation
HETEROLYTIC (CHARGE HAS MOVED!)
DESCRIBE ALPHA CLEAVAGE
So our radical has a tendency to pair up (electron pairing) SO can cause another single electron from a non adjacent bond to join it and form a double bond -which means the electron that was left behind stays on the cleavage product
HOMOLYTIC - charge stays (even electron ion) - cleaved product has the radical now (neutral radical)
our original keeps the charge
WHO WINS OUT BETWEEN INDUCTIVE AND ALPHA CLEAVAGES
inductive cleavage is a lot more likely when the atom holding our dot cation is LARGE electronegative - so when we split it retains the electorns and radical and the deficiency (the charge) is on the other guy (makes sense)
ORDER for this
halogens > R , pi, S, O > N
reverse order for ALPHA CLEAVAGES
these favor those that are good electron donors
everything else prefers inductive?
So N want to alpha cleave and donate electrons
while halogens want to inductively cleave
but both can do both and we will see on spectra
Draw mclafferty rearrangment and what are the requirements (also what unique type is produced here0
radical cations, unsaturated functional groups, 6 emmbered intermediate - includes H, creates distonic radical cation (RADICAL ODD ELECTRON _ unique - because we rearranged)
EI vs soft ionization
EI makes Odd electron ions which can make EE cations (with neutral radicals) or Odd electron cations (radical) with a neutral
WHEREAS soft ionization simply makes even electron ions to begin which fragment to make more even electron products and neutral
strategies for EI analysis
nitrogen rule
double bond rule (2*c + 2 +zN - H - Y ) /2 = number of double bonds
Ou electron is going to be removed from lone pair, then double bond , then sigma bond
ALPHA and INDUCTIVE cleavages leave the charge on different spots - inductive is heterolytic and alpha is hemolytic so if we can get the mass of a frag - we can tell how it fragmented because it must have the charge!
STEVENSON rule for whats likely (abundance of ions)
Why is metabolomics good?
we’re diverse so metabalome reflects taht (eg genetics, age, environment, disease, nutrition, drugs - metabolism TIES everything together - proteins , lipids etc
ANABOLISM vs CATABOLISM
anabolism is synthesis of molecules, catabolism is breaking down
Why can metabolomics be difficult
There are known pathways that make metabolites bUT A LOT OF UNKNOWNS
What is THE METABOLOME
collection of small molecules in tissue, plasma, urine and tissue
DEFINE TERMS:
global metabolomics
metabonomics
targeted metabolomics
Global i suntargeted - measure as many as can - semi quant
Metabanomics - referred to NMR based metabalomics
targeted - measures select group - quantitatively - triple quad
Metabolites can be divided into 3 categories: what
Enzyme products (canonical metabolmics), things we take in EXPOSOME and the those from chemical reactions (non enzymatic)- REACTOME !
METABOLOMIC WORKFLOW
sample collection (tissues etc, disease control), sampe prep (homogenization , ISTD< extract , protein precip etc);
SAMPLE ANALYSIS _ NMR MS
Data processing - stats, extraction
Biomarker ID - ID/quant
How does HRMS play into metabolomics
HRMS - for ID
intensity for concentration
ID can also use - RT, m/z match, MS/MS
ALIGNMENT in metabalomics
alginmed peaks integrated together for peak height or area - don’t include isomers!
METABOLITE CONFIDENCE LEVELS!
LEVEL 1 - ID by 2 independant orthogonal pieces of data compared to a standard on same conditions (eg RT and m/z
LEVEL 2 - putative annotation - ID without reference but similar to library
LEVEL 3 - Putative characterization - ID based on similar chemical species
LEVEL 4 - not known
Metabolomic study objectives
1 - biomarkers
2 - unknowns
3 disease pathogenesis
4 - metabolic regulation (isotopes needed0
Challenges of metabolmics
false discoery rates,
no reference material for + metabolites
metabolite ID
metabolite cannot be identified
fragmentation can be unpredictable
many metabolites are common so need to discern biological source
also chemical and structural diversity - not a class like lipids or proteins ( 50 -1500 DA,
so extraction can be difficult (also ionization etc analytical performance
Dynamic range - concentrations vary pM to mM
Metabalomic strengths
many are common across species, dynamics are fast, and sample prep can be simple or complex depending on goals
Issues with chroamtography in metabolomics
RP - highly poalr not retained - so void volume can have a lot of species
- ion pair reagents can help but can dirty MS
HILIC - lipids not retained
Issues with detection in metabalomics
so mayn types not one method gets them all
Targetd vs untargeted metabolomics
targeted - QQQ
untargetd QTOF or orbitrao
targeted is easier
untargeted is a lot of data - and a lot of that diversity
targeted has specified extraction etc
What is translating the analysis
Basically moving a developed method to clincial sample test - health is a continuum can you distinguish your markers from other markers or other disease states
In complex formation what determines the Ke REL that ends up in precurosr
Complex DOF,
fraction of DOF from complex in ion,
Complex lifetime (which depends on complex well depth, complex DOC and decreases with Ke rel)
What types of things like to form a complex (T-V)
highly poalrizable targets
LOW KE rel -
high efficiency Q/Ke rel
large b causes a negative deflection
Characteristics of binary - collision (T->V)
Timing sensitive
caused by rapid change in polarization
vound to elastic limit
LARGE B, - little scattering
small energy transfer
not common
What are some characteristics of impulsive colision (BINARY)
widely applicable - AN ELASTIC COLLISION with part of the ion
Positive deflection (small b)
LOW efficiency -
one collision
tends to have higher Kerel
INDEPENDANT OF COLLISION TIME!
Vertical Excitation describe
Large B- scattering
Not likely
interaction times less than 10-14 s?
Generally high E (generally not favorable
Curve Crossing describe
Most LIKELY for T-? E
slow enough for transition to upper state, but fast enough to cross back down to original state
prefers SMALLER velocities E and b
scattering determined by collision energy but generally associated with fast collisions
Mechanism T-> EV curve crossing + IMPULS COLLISION
direct vibrational excitation with electronic transition
So curve cross and then impulsive collide - - long and short times
List all the mechanisms of activation (translation to ..)
What are the things you shoudl ask yourself
KeREl
b
scattering
Timescale
Q? maybe
likelihood
energy type
Draw an ninteraction potential
In slow heating ctviation energy diagram what do the rate steps determine
SO if dissociation is Rate limitng - we see a normal boltzmann distribution - this is higher pressure FAST energy exchange
If activation is rate limiting - then we get truncated botlzmann - (low pressure)
Write out Lindemanns ideas
do it
Its essentially write out the equations
add them all up set to 0 based on steady state
solve for A*
then if K1»_space; K2 you can get rid of it and the reaction becomes first order (HIGH PRESSURE)
IF K2 > K1 - it stays second order (low pressure) which makes sense - high pressure would mean more actiation
What does lindemanns theory not tak einto account
INTERNAL DOF - already possess some energy
Doesnt account for energy level dependence of reaction rates (at higher pressures his rates fail)
doesn’t take into account bond specific where reaction takes place
What did hinshelwood add and whats the strong collision approximation
Takes into accoutnt DOF -
- did this by probability of a reactants level of activation is a boltzmann distribution - so this accounts for internal motion -
AS such the strong collision approximation states - if we assume the energy of a molecule is randomized according to the boltzmann dist - then the ACTIVATION rate is proportional to the boltzmann dist as well
Problems with hinshelwood
Only addresses energy of activation (half of the reaction) K2 is assumed to be the same which presumably isn’t true
K1 has a dependence on Number of frequencies but K2 doesn’t here of the mode of interest
Assumes all oscialltors are effective they are not
What did RRK add
it added 2 things
1) distinction betwen energized and activated molecule
Energized has the energy to become activated but needs to vibrate more
ACtivated ready to dissociate
2) They also added a model for degeneracy (how many ways j objects can fit among s boxes) ( can see the equations on slide) 0 which can be then used for the rate (comes down to
((E - E) / E) ^ (s-1) ( the probability of locating in the dissoc mode a minimum amount of energy E out of the total energy E) - so its the probability that one of these oscillators has our E* energy
Take aways from RRK
K2 increases with E because the probabiltiy of localizing E0 goes up
K2 decreases as S goes up (# of boxes - because more modes to spread energy - this is incontrast to K1)
What was RRK;s issue
statistics of coupled oscillator is vague
assigned k double dagger first order frequency - doesn’t account for reactions with higher frequency factors (only those with average vibrational frequencies)
What did RRKM add
Transition state theory - we can get our rate by determining the # that just cross the transition state barrier by the total number of states
Individual vibrational frequencies of energized molecules considered explicity
RRKM assumptions
time for dissoc is long compared to form, excite, activate
Time for dissoc is long relative to energy distribution (ONLY APPLIES TO ERGODIC PROCESSES)
- adiabatic - only on one energy surface
Products are from competing and consecutive reactions
TS - once past noo return
In a mclafferty what are the two products gonna be
ODD ELECTRON (radical) cation) an d aneutral!!
Steps for protein spectra analysis
1) Look for immonium ions (low mass range)
2) look for B2 ion ( a mix of 2 ) (and get y n-2 ion)
3) Look for y1 - if tryptic - 147 lysine or 175 arginine
4) ID y n-1 ion high mass
5) Extend y series
6) extend b series
7 look for water loss, NH3 loss, HSOCH3 loss etc
RRKM EQUATION
K(E) = reaction path degeneracy (# of ways) ; Sum of states at transition state energy )
and inversely related to density of states at energy E (ways to not react) and planks constant
Native vs non native spectra - major differences
non native ESI spectra - A LOT OF PEKAS - native will have bigger/shaggier but less
What are some things to look for in native MS spectra
Bound vs unbound ( so I guess a change in mass but not of charge - BUT also - potentially a change in conformation, MUltiple charge states
LOSS of units (eg tetramer to monomer - big loss of MASS and CHARGE
