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