what can proteomics do for you? · 2018. 12. 5. · (dynamic range) resolution costs sensitivity:...
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What can proteomics do for you?
Maarten Dhaenens
Belgian Proteomics Association
DNA RNA Protein
The Machinery of Life, D. Goodsell
Data Paper
Abbreviations in the final slides
Studying Proteins
“Exploring” the proteome
“PinPointing” the proteinsWhere are these proteins present?
Antibody-based approaches:• Western Blot: detect presence
• Flow cytometry: detect cells expressing the protein• Microscopy/immunohistochemistry: where in the cell/tissue?
MS approaches:• Multi-reaction Monitoring (MRM): very high sensitivity detection of targets
• Affinity Purificartion MS (AP-MS): protein interactions• Mass spectrometry imaging (MSI): where in a tissue?
“Imaging” the proteinsHow do these proteins look like?
X-ray crystalographyCryo EM
MS approaches:• Native MS (using Ion mobility Separation)
• Cross-linking MS • HDX-MS
• …
Explore
Detect
Image
Identifying the proteins of a proteome• Edman Degradation• Nanopore sequencing?• MS
• Top-down• Middle-down• Bottom-up
Quantifying proteins of a proteome• 2D PAGE/DIGE (gel electrophoresis)• MS
• Label-based• Label-free
Which proteins are present and how much of them?
https://belgianproteomics.be/expertise
MS-based Proteomics (MSP) = hypothesis generator
Explore
Sample Preparation(Francis Impens
& Baptiste Leroy)
Identification(Lennart Martens)
Quantification(Sebastien Carpentier)
Trypsin
Acquisition(Maarten Dhaenens)
“Bottom-up” Proteomics
K
R
http://blog.nonlinear.com/2018/03/28/proteomics-peptides-emergence/
PTM
MSP in 2015: Six theses from a (grumpy?) old man
“All the proteins all the time” – a comment on visions, claims, and wording in mass-spectrometry-based proteomicsWolf D. Lehman, Anal Bioanal Chem (2015) 407:2659-2663
Defining Proteomics as a progressionof genomics is disputable
Mass spectrometry-basedproteomics leads to a cult of
unprocessed data
Streamlined wording in MSP creates an “all problems solved”
impression
Error reduction in MSP requiresimproved bioinformatics, not
manual curation
Quantitative MSP is also error-prone, because it is a hybrid of MSP
and standard quantificationmethods
In MSP a marginal analytical coverageis sufficient for an “almost complete
picture of the proteome” (1p2h)
Genomics = sequencing
DNA RNA
Peptide
Proteomics = Weighing
Protein
“Weighing” the proteins of a proteome
• 1D/2D Polyacrylamide gel: 0,1 kDa resolution• Mass spectrometry (MS): 1mDa resolution
Data Analysis in Quantitative Proteomics:Framing a Fuzzy Picture
BIG N2N Seminar 2015:
“Nano” flow = 300nl/min“Micro” flow = 5 µl/min
LC-MS: an invaluable asset to the field, but a spirited one
nanoLC ESI Q-TOF
(Vacuum)
0.1% FA (H+)
Charge 1+-x+
Capillary withvoltage applied
N2 flow forevaporation
In source decay
Differential ionization efficiencies
Ion Suppression
Partial sampling
Charge state distribution
VacuumPS: Tryptic peptides are 2+-x+,
partially because of basic terminal R or K
ESI: Electrospray Ionization
Alternative Ionization = MALDI 1+(matrix-assisted Laser Dissorption Ionization)
m/z
Gaussian transmission efficiency
Chaotic fragmentation
Co-selection leading tochimericy
“Neutral loss” of PTM
Loss of Neutral Fragments
Positively charged only
(Inert gas)
Q: Quadrupole
CID: “Collision-induced Dissociation”HCD: “Higher Energy Collisional Dissociation”
Limited duty cycle
CID/HCDDetector saturation
(Dynamic range)
Resolution costs sensitivity: ion beam “skimming”
Or W-mode
Mass (m/z) Analyzer: TOF: Time-of-Flight
Arginine or
Lysine
H
y2
b1 b2
y1H+
Mass (m/z) Analyzer: Orbitrap
Resolution costs sensitivity: Longer scan time
Detector saturation(Dynamic range)
Limited duty cycle
nanoLC ESI Q-Exactive
Trapping allows parallellisation
Trapping allows parallellisation
E.g. MSMSMS or MS3 to fragment fragments:• Backbone sequence following neutral loss• Increased quantitative accuracy of TMT
• …
Atom: nucleus + Electrons
Nucleus:Protons + neutrons
Protonsand neutrons:
Quarks and gluons
Naturally occuring isotopes: e.g. Carbon
Mass (m/z) Analyzer: Accuracy
1.67 × 10-27 kilograms = 1Da
1 additional neutron
2 additional neutrons
Charge can be determinedbased on the m/z differencebetween isotopes of the same peptide:1 = 1+0,5 = 2+0,33 = 3+0,25 = 4+0,2 = 5+…
The use of isotopes:Label-based Quantitation
8 additional neutrons
Peptide (= e.g. C56H123O14N12S2)
m/z
m/z
ACQUISITION
Data Dependent AcquisitionDDA
…The Convention
MS scan = “Survey scan”:quadrupole passes all incoming ions
MSMS scan = fragmenation spectrum precursor selected by quadrupole
Acquisition: DDA (Data dependent analysis)
Trypsin
Arginine+
Lysine+
Interesting precursors in the MS scan=
Highest abundance & Multiply charged.
MS: m/z
LC-MSMS DDA RUN:2D representation
LC: RT
ONLY 20-50% of peptide-like precursors
get selected!
90min
DDA Identification
From: PhD Disseration of Giulia Gonnelli
Machine learning is here to stay
Only 20-40% of MSMS spectra get annotated
DDA Proteome coverage
In MSP a marginal analytical coverageis sufficient for an “almost complete
picture of the proteome” (1p2h)
Defining Proteomics as a progression of genomics is
disputable
blog.nonlinear.com/2018/03/28/proteomics-peptides-emergence/
MS: m/z
RT
m/z
Intensity
XIC
!AUC
DDA Quantification: Intensity
LC: RT
Quantification: Technical Variation Accumulates!
Bantsheff et al., 2012“Quantitative mass
spectrometry in proteomics”
Anal Bioanal Chem
! Amino acidscan be metabolically
converted!
Trypsin @ K and R
Label-based Relative Quantification: SILAC = Metabolic incorporation
Label-based Relative Quantification: iTRAQ/TMT = Chemical Labeling
iTRAQ TMT! Instrument requirements!
“Different peptides have different ionization efficiencies”
AUC ≠ Number of ions present in the sample
Label-based Absolute Quantification : AQUA (Absolute QUAntitation): Spike-in
! Limited number of targets!!Assay optimization!
“Label-free” Relative Quantification =Precursor-based bottom-up label-free
Precursor-based = MS1-based
A list of significantlyup- or downregulatedtargets is generated
!Experimental design!= Sample Prep= Acquisition
= Data Analysis
! Technical variation accumulates!!Batch effects!
Target Confirmation: MRM(on a TripleQuad instrument or QQQ)
Precursor + Fragment XIC = MS2-based(=Transitions/Traces)
PRM = HR-MRM = DDA repeated over RT on selected candidates (Q-Orbi/Q-TOF instruments)
(AQUA)
(HR-)MRM or PRM Target Confirmation
Label-free MS1 Quantification
Generating Targets and confirming them:
ACQUISITION
Data Independent AcquisitionDIA…
The Paradigm Shift
!
LC: RT
MS: m/z
HDMSESWATHor AIF
DDA DIA
LC/MS
CELow CEHigh
Q1 Transmission w
indow
MS MSMS
TripleTOF (Sciex)
SWATH = Quant Only
“MRM-like post-acquisition data extraction”
Build the (extended) library once :2DLC, gas phase fractionation,…
XIC AUC
XIC AUC
Identification < DDA library
HDMSE = Quant and ID
SynaptG2Si (Waters)
HD = High Defenition = using “Ion Mobility Separation”
“Ion Mobility”?“Shape” (CCS or Ω) of the molecule TOGETHER WITH the
gas determines Drift Time (dt)
T-Wave
“What is the size of an ion?” = “What is the sound of one hand clapping?”
DDA
ID Quant
SWATH
ID Quant
HDMSE
ID Quant
Data Dependent Acquisition (DDA) Data Independent Acquisition (DIA)
No
MSM
SM
SMS
x%
x%
Frag
men
t “Al
l”
Data Analysis
Proteomics
Explore
Confident resultsare real
Doubt:False Positives
Doubt:False Negatives
Wrong result:Wasted time and money
Missing ValuesCourtosy:
Progenesis(Waters)
Appendix
Abbreviations & DefenitionsLC/MS Challenges and Solutions
TMT labels
Abbreviations & Defenitions
Technical Terms
ESI Electrospray Ionization Oft-used ionization technique in proteomics, mostly directly coupled to LC (generates all charges)
MALDI Matrix-Assisted Laser Disssorption Ion… Ionization technique wherein sample is spotted onto a plate in a matrix (generates 1+ ions only)
LC Luiquid Chromatography Separation technique to separate precursor peptides in time
RT Retention time Time where a specific peptide elutes from the LC column
XIC Extracted Ion Chromatogram Intensity profile of a specific precursor eluting from the LC system
AUC Area Under the Curve Is proportional to the amount of a precursor present in the sample (relative!)
TOF Time-of-Flight Method for measuring m/z (counterpart of the Orbitrap)
CID Collision-Induced Dissociation Fragmentation technique that breaks peptides by colliding them with inert gas (Ar or N2)
HCD Higher Energy Collisional Dissociation CID-like fragmentation method with higher energy, implemented on orbitrap instruments
IMS Ion Mobility Speration Separates molecules based on their “shape” (CCS)
T-wave Travelling Wave Specific type of IMS that is build into the Synapt series of mass spectrometers
dt drift time Unit used to define differences in CCS (equivalent to m/z in mass spectrometry)
CCS Collisional Cross Section Unit of “shape” rather than mass of a molecule (as defined by IMS)
Chimericy When two precursors of similar m/z are co-selected and co-fragmented into a single MSMS spectrum
Acquisition strategies
DDA Data-dependent acquisition MS scan is used to define interesting precursors for selection and fragmentation
DIA Data-independent acquisition All ions (or a specific window) are isolated and fragmented together
HDMSE High Defenition MSE DIA strategy allowing for both ID and Quant (no quadrupole selection)
SWATH Sequential Window Acquisition of allTheoretical masses
DIA strategy that sequentially acquires mass windows, allowing for quantification of “all” fragmentsbased on a prior DDA library (Quadrupole selection)
AIF All Ion Fragmentation SWATH-like acquisition on an orbitrap instrument
MRM Multi Reaction Monitoring Targeted MS approach quantifying a specific preset of precursors on TripleQuad)
PRM Parallel Reaction Monitoring Targeted MS approach quantifying a specific preset of precursors on Q-TOF or Q-Orbi instruments
MS3 MSMSMS Selecting fragment ions in MSMS for one more round of fragmentation
Abbreviations & Defenitions
Biology
PTM Posttranslation Modification Chemical modification on proteins that change their activity (E.g. Phosphorylation)
K Lysine Terminal, basic and thus charged amino acid in tryptic peptides
R Arginine Terminal, basic and thus charged amino acid in tryptic peptides
In source decay
Differential ionization efficiencies
Ion Suppression
Partial sampling
Charge state distribution
Gaussian transmission efficiency
Co-selection and chimericy
Chaotic fragmentation
Neutral losses of PTM
Loss of Neutral Fragments
Detector saturation
Limited duty cycle
Resolution costs sensitivity
Methodology Acquisition Bioinformatics
Inlet (e.g. StepWave)
Machine learningModeling
New search algorithmsProcessing
(e.g. peak picking)…
Quan: Label-based CE instead of LC
Quan: Label-based CE instead of LC
Source parameters DIA: HDMSE
LC Buffers/ESI gas DIA: HDMSE
Quad design (SWATH)
Increased (2D)LC separation Quad resolution
Label-based ID Other, such as ETD,…
Enrichment/Derivatization ETD, MS3,…
Depletion protocols DRE, ADC detector,…
DIA, Orbitrap, WE,…
Orbitrap
BETTER IDENTIFICATION and QUANTIFICATION
ESI
Quad
CID
TOF
LC/MS Challenges and solutions
TMT / iTRAQ