Post on 22-Dec-2015
Mass spectrometry-based proteomics
Jeff JohnsonFeb 19, 2014
MS Proteomics in a Nutshell
• Ionization– Delivering macromolecules to the MS
• Ion Manipulation– Trapping and ejecting analytes of interest
• Fragmentation– Breaking apart for more information
• Mass analysis and detection– Measuring masses and quantifying intensities
MS Proteomics in a Nutshell
• Ionization– Delivering macromolecules to the MS
• Ion Manipulation– Trapping and ejecting analytes of interest
• Fragmentation– Breaking apart for more information
• Mass analysis and detection– Measuring masses and quantifying intensities
Macromolecular Ionization for MS
• Analyte must be in the gas phase for mass analysis• Analyte must be charged in order to be
manipulated by electric and magnetic fields– All mass analyzers measure mass-to-charge ratios (m/z)
• Two predominant approaches (shared the Nobel prize in 2002)– Matrix assisted laser desorption ionization– Electrospray ionization
MALDI Ionization• Sample is spotted in a matrix that
readily absorbs UV/IR light and is vaporized by a laser– Common matrix: 2,5-dihydroxybenzoic
acid (DHB) • Advantages
– Fast and easy– Spots can be reanalyzed later– Most analytes get one +ive charge
makes it easy to deconvolute• Disadvantages
– Harsh. Often breaks analytes apart (e.g., breaks phosphorylation)
– Not easily combined with online HPLC separations
Electrospray Ionization• Sample is dissolved in liquid and pushed through
a charged needle and sprayed into an evaporation chamber
– Droplets pulled into the MS source by electric potential between the needle and the MS
– Heated ion transfer tube evaporates water molecules in droplets leaving +ively charged analytes in the gas phase
• Advantages– Compatible with online HPLC separations– “Soft” ionization maintains label and non-covalent
interactions• Disadvantages
– Analytes can have different numbers of charges, can be difficult to deconvolute without high mass accuracy
– Different samples going through the same electrospray tip causes carryover problems
• Especially bad with online HPLCs
Ionization is Nearly Impossible to Predict
2x
A B
A B
X
• Different molecules ionize with different efficiencies and are very difficult to predict
• MS intensity ratios between different molecules do not reflect ratios in the sample from which they were derived
• Most quantification by MS is relative
Ionization is Nearly Impossible to Predict
• Different molecules ionize with different efficiencies and are very difficult to predict
• MS intensity ratios between different molecules do not reflect ratios in the sample from which they were derived
• Most quantification by MS is relative
A A
A A
Sample 1 Sample 2
Sample 1 Sample 2
* Assumption: MS run 1 = MS run 2
MS Proteomics in a Nutshell
• Ionization– Delivering macromolecules to the MS
• Ion Manipulation– Trapping and ejecting analytes of interest
• Fragmentation– Breaking apart for more information
• Mass analysis and detection– Measuring masses and quantifying intensities
Ion Manipulation
• We need a way to select only ions of interest– Most detectors are just electron multipliers that don’t
measure mass but just detect a thing hitting the multiplier– We can manipulate ions to deliver defined mass ranges to
the detector to get a mass spectrum• Two common tools:
– Ion traps– Quadrupoles– Both use electric and magnetic fields to select ions of a
particular m/z range
Ion Trap
• Ions are trapped by 3D electric field by DC and AC applied to the electrodes
• An ion trap can accumulate ions as they come in from the source and store them
• Low resolution: +/- 1 Da
Quadrupole
• Can be thought as a mass filter• DC and AC fields applied that stabilize a trajectory for ions in
a desired mass range, undesired ions are ejected• Quadrupole operate with a continuous flow of ions• Low resolution (+/- 1 Da)
MS Proteomics in a Nutshell
• Ionization– Delivering macromolecules to the MS
• Ion Manipulation– Trapping and ejecting analytes of interest
• Fragmentation– Breaking apart for more information
• Mass analysis and detection– Measuring masses and quantifying intensities
Fragmentation
• Usually measuring the mass of an analyte is not enough to conclusively identify it
• By fragmenting an analyte and measuring the masses of the fragments we can obtain further information to identify the analyte
• There are many types of fragmentation but collision-induced dissociation (CID) is the most common– Fastest and most generally successful for the widest
variety of proteins and peptides
Collision-Induced Dissociation
• Give ions kinetic energy and collide with gas molecules (He)• Collisions build up potential energy until a fragmentation
event can occur• Ideally potential energy is strong enough to break a single
peptide bond but not strong enough to fragment further• Can be done in an ion trap or a quadrupole
Collision Induced Dissociation
A E P T I R H2O
Fragment (somewhat) randomly along the peptide backbone
M/z
Inte
nsity
A E P
A
A E
A E P T
72.0201.1
298.1399.2
B-type Ions
M/z
Inte
nsity
R I T P E AH2O
Y-type Ions
M/z
Inte
nsity
R I T P E AH2O
B-type, A-type, Y-type Ions
MS Proteomics in a Nutshell
• Ionization– Delivering macromolecules to the MS
• Ion Manipulation– Trapping and ejecting analytes of interest
• Fragmentation– Breaking apart for more information
• Mass analysis and detection– Measuring masses and quantifying intensities
Mass Analysis and Detection
Magnetic Sector MS
• All mass analyzers achieve the same thing: physical separation based on mass:charge
• Magnetic sector is the simplest and one of the earliest types
FT-ICR MS
• FT-ICR = Fourier transform – ion cyclotron resonance• Ion injected in line with a strong magnetic field that
induces a cyclical motion• Radius of the cyclotron motion is proportional to m/z
Time-of-flight MS
Medium / High Resolution
Quadrupole and Ion Trap MS
Electron multiplier
• You can use a quadrupoles or ion traps to “scan out” ions across an entire mass range to a detector by gradually ramping voltages
• Low resolution but electron multipliers make these very sensitive
Orbitrap MS
22
R
Rmzr
12
2
R
Rmz
zm
kz /
• Characteristic frequencies:– Frequency of rotation ωφ
– Frequency of radial oscillations ωr
– Frequency of axial oscillations ωz
r
)/ln(2/2
),( 222mm RrRrz
kzrU
z
φ
Power of Fourier Transforms
• FTs convert from time domain to freq domain
• Instead of a single measurement the m/z is measured over a period of time and the FT essentially averages all those measurements
• Resulting data is very high resolution
Chromatography to Simplify Complexity
• Complexity hurts sensitivity• A constant, defined number of ions can be analyzed in
each MS scan• Sensitivity is constant (around 1 fmol)• A scan with fewer ions is more sensitive than a scan with
many
ComplexSample
MS
Chromatography to Simplify Complexity
C18 RP column
ACN gradient
AB
C
D
A
B
C
D
Complex Sample HPLC MS
Chromatography to Simplify Complexity
VeryComplexSample
Online HPLC (RP) MS
Offline HPLC(e.g., SCX) SCX Fractions
SCX FractionsInjected individually
Acquiring MS Data
• Data acquisition depends on experimental goals– Data-dependent acquisition
• MS attempts to acquire data to allow you to identify a maximum number of unknowns
• Commonly used for analyses where you don’t know what you’re looking for
– Targeted acquisition• MS only acquires data for what you tell it to acquire• Much more sensitive than data-dependent, but also more
limited in scope
Data-Dependent Acquisition
Data-Dependent Acquisition
High resolution survey scan(<5 ppm mass accuracy)
1
23
Data-Dependent Acquisition
Low resolution MS/MS scan 1
Data-Dependent Acquisition
Low resolution MS/MS scan 2
Data-Dependent Acquisition
Low resolution MS/MS scan 3
Peptide IdentificationAA sequence DB(Species UniProt)
Peptide Identification
1
2
3
AA DBsrestrictedby parent ion mass measured in survey scan
AA sequence DB(Species UniProt)
Peptide Identification
MS/MS 1
MS/MS 2
MS/MS 3
1
2
3
AA DBsrestrictedby parent ion mass measured in survey scan
AA sequence DB(Species UniProt)
Probabilistic Matching (X!Tandem)
by-Score= Sum of intensities of peaks matchingB-type or Y-type ions
HyperScore=Hyper Score
# of
Mat
ches
Best HitSecond
Best
Model spectrum comparisons
Pattern Matching (Sequest)
Sequest XCorr
Cross Correlation(direct comparison)
Auto Correlation(background)
XCorr =
Offset (AMU)
Corr
elati
on S
core
Targeted Acquisition with a QQQ
A priori knowledge required:SRM assay development for a list of proteins/peptides of interest Information derived from label-free unbiased
proteomic analysis
SRM Assay
“Sensitivity”• Sensitivity of a MS is well defined, but the ability to
identify something is a very different concept– Ability to detect depends on:
• Sample complexity• MS sensitivity• MS speed
– A faster MS can collect go deeper in each survey scan– Think “top 10” vs. “top 50”
• MS mass accuracy– Better mass accuracy improves the ability to identify peptides but
sacrifices speed and MS sensitivity– Especially important for variable modifications
– The “best” method is very dependent on the experimental goals
Database SearchingIon trap+/- 1 Da
Orbitrap+/- 0.002 Da
Database“search space”
Database SearchingIon trap+/- 1 Da
Orbitrap+/- 0.002 Da
Database“search space”
+S/T/Y phosphorylation
Database SearchingIon trap+/- 1 Da
Orbitrap+/- 0.002 Da
Database“search space”
+S/T/Y phosphorylation
Protein Quantification
• A mass spectrometer is an inherently quantitative device but the ionization source is not– Different peptides/proteins are ionized with drastically
different efficiencies– Absolute abundances in a mass spectrometer are not
precisely indicative of abundance in a sample• Solution: stable isotope labeling
– Compare samples that have been labeled with stable isotopes (13C, 15N, 2H)
– ‘Heavy’ isotopes behave chemically identically to their ‘light’ counterparts but are separated in the MS
Isotope Coded Affinity Tag (ICAT)
Stable Isotope Labeling of Amino Acids in Culture (SILAC)
• Grow cells in media supplemented with stable isotope-labeled amino acids
• Combine samples at the level of cells and process as one sample• Minimize variability
between samples for lysis and digestion
• Different samples separated by mass in the MS
Absolute Quantification (AQUA)
Absolute Quantification (AQUA)