ADVANCING BOTTOM UP PROTEOMICSWITH COVARIS Your samples deserve the best treatment!

APAC | September 9 th 2020 | www.covaris.com

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“We have tested this specific platform

extensively in our laboratory at the Max

Planck Institute of Biochemistry,

Munich. Using our materials and setup,

we can now prepare hundreds of

biological and clinical samples in a

highly streamlined manner.”

Professor Matthias Mann

Director at the Max Planck Institute of

Biochemistry

Head of the Clinical Proteomics group at the Novo

Nordisk Center for Protein Research

2

• Our Business

• The Science Behind the Scene

• Our Solutions

• Results

Agenda

3

Mission

Accelerate discoveries in bio-analysis with cutting-edge

pre-analytical sample prep solutions

Goals

• Increase results quality

• Revolutionize high-throughput bio-analytical

applications

• Set new standards

• Clinical sample prep

• Cell Lysis

• Biomolecules Extraction

• Formulation

Sample preparation

Acoustic

Physics

Molecular

Biology

Mechanical

Engineering

Biochemistry

Automation

Software

development

Covaris at a Glance

Sample Prep

4

Over 6,000 AFA systems installed in 30+ countries

11,000+ peer-reviewed articles referencing Covaris (+200 per month)

Our Customers

Broad market acceptance

5

The Covaris Product Line

Pre-Analytical Sample Prep Instrumentation

Large choice of instruments and consumables for any level of throughput and automation

cryoPREP® Dry Impactor

Tissues and other difficult Samples Prep

In-line AFA system

for Pilot and Large-Scale

Production Formulation

Adaptive Focused Acoustics® (AFA®)

New workflows for high molecular weight nucleic acids

extraction

NGS shearing Gold Standard and More!

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cryoPREP dry-pulverization enables ideal biomolecule extraction

Fast processing/no heating/contact-free/reproducible/easy/clean/safe

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Covaris Focused-ultrasonicators

+

S-series• Single sample

processing• High power

M-series• Single sample

processing• Low to medium

power

L-series• Processes up to 96 samples

per run• parallel process, 8X faster,

compatible with robotization• Medium power

Evo-series• Processes 1-8

samples per run• High power• Upgrade to E

ME-series• Single to 1-8

samplesprocessing

• Low to medium power

ME/ML/L series : Compatible with AFATUBE- 10 µL to ~150µL Allows single pot workflows

S/E series for High power applications :o Difficult/higher amount of tissue or species

(Yeast, plant,…)o Formulation

Compatible with bigger volumes (2-20mL)

All instruments : DNA, RNA, Chromatin shearing/Cell lysis/Soft tissue lysis/NA extraction/protein extraction/protein processing

available in 15µL/50µL/130µL/0,5mL

ML-series• Single to 1-8

samples• parallel

processing• Medium power

E-series• Processes 1-96

samples per run• High power• Upgrade to LE+

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• Our Business

• The Science Behind the Scene

• Our Solutions

• Results

Agenda

9

Extracorporeal Shockwave Lithotripsy (ESWL)

• Apply non-contact acoustic properties to break down kidney and gall stone

Well-established medical technology

• Precise

• Reliable

• Controlled

SamplePatient

Adaptive Focused Acoustics® (AFA®)

Covaris Technology – The Origin

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Covaris systems operate in ultrasonic range 𝑯𝒊𝒈𝒉 Frequency (Hz) =speed of sound in water𝑺𝒎𝒂𝒍𝒍 Wavelength (mm)

High frequencies ultrasounds generate small wavelength for a precisely localized process

Covaris – The Wavelength Secret

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AFA – A technology designed to fully control biological sample processescomputer-controlled electric input to fine tune acoustic energy

Adaptive Focused Acoustics (AFA) System80+ Patents Granted and Pending

Optimized

ConsumablesComputer

Control

Sensors

Adaptive

Energy input

Controlled

Environment Focused

Acoustics

Control

• Intensity

• Cycles

• Time

SCHEMATIC OF RELATIVE SONICATION ZONES

Kept in a cooledwaterbath

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Covaris system:

• Less energy

• Maintain isothermal process

• Transducer geometry

• Small wavelength

• AFA optimized tube

Lateral pressure field at the

focal point of the transducer

Acoustic field shaped to penetrate

optimized biological sample vessels

Control of Focal Zones

Energy needed to reach cavitation threshold (1MPa)

AFA

Non-contact

0.8 Watt

Probe Sonicator

(sample contact)

4.6 Watt

Bath Sonicator

(not focused)

130 Watt

Same process - end point

Covaris

microTUBE

2 MPa at 180s

Bath Sonicator

microfuge

2 MPa at 600s

Probe Sonicator

microfuge

2 MPa at 300s

No heat

accumulation

On target

The Secret of FocusingLarger sonication zone = higher energy required =heat

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Parallel processing Process precision

Wide range of applications - Ready for the next challenge

AFA in Action

Mixing Carbon nanotubes resuspension Cell pellet dissociationFFPE emulsification Fast on-demand thawing

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• Our Business

• The Science Behind the Scene

• Our Solutions

• Results

Agenda

15

Adapting the process to the sample

(…not the opposite)

Delivering the right dose of energy optimized for maximum performance at each step of the process

Simple

Dry CryofractureDisruption Homogenization Lysis Shearing

Purification

CleaningDigestion Resuspension

cryoPREP

AFA

Large volume

Frozen tissues…

Fibrous tissues,

Yeast, FFPE…Soft and/or

delicate tissues…Cells, bacteria…

Covaris Science of Sample Preparation

A Streamlined Workflow

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The possibilities offered by AFA for proteomics

Add

reagents

for

digestion

Cell Lysis

and

Protein

Extraction

Dispense

sample in

plate or tube

Enhance

Digestion

with AFAAdd

buffer

of

choice

Add

magnetic

beads for

clean-up

Improve

washing with

AFA

micromixing

Improve

peptides

resolubilization

after elution

Sample types :Cells (Human, animals, plants, yeast, bacteria…)Fresh tissueFormalin Fixed Paraffine Embedded (FFPE) tissueDBS…

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Representation of 6 On-deck R230 systems

combined with a liquid handler and Robot

LE220R-plus solution, using a Robotic

arm and Liquid handler

Scalable and Easy Implementation

Automation with Robotic Arm

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Continuous developments and improvement

Simultaneous processingFrom strips to 500ul tubesCompact footprint

384 for throughputOptimized kinetics for low volumesLow bind

Full robotic integrationLarge market compatibility8 strips to 384

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• Our Business

• The Science Behind the Scene

• Our Solutions

• Results

Agenda

20

0

Starting with a universal protocol particularly well suited for FFPE samples

• >200% protein of “standard” protocol

• Near identical ID rates to fresh frozen

Wilson, Wojcik et al. - US HUPO 2019 posterWilson, Pappin et al. - HUPO 2018 poster

• Protocol is universal• More proteins are extracted• No extraction bias

Marchione, Wojcik et al. – J Prot Research 2020

“HYPER-sol: flash-frozen results from archival FFPE tissue for clinical proteomics”

• Correlations between fresh frozen and FFPE (direct) are very good -> more uniform and complete extraction

• Old samples can be processed with the same quality

“Total solubilization of FFPE samples for high throughput clinical proteomics”

“Universal Sample Processing Of Multiple Sample Types For Reproducible Proteomic Sample Preparation”

with

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Bottlenecks in current proteomics sample preparation protocols

➢ Addition of detergents to cell lysis buffers is problematic

➢ Multiple sample handling steps for protein/peptide clean-up and digestion

➢ Multiple handling steps introduce variability

➢ Peptide recovery based on filtration, centrifugation, re-solubilization causes sample loss

➢ Process takes long

➢ Parallelization/automation is difficult

Adapted, courtesy of Jeroen Krijgsveld, DKFZ Heidelberg

Vienna ESCP conference, Aug 2019

• Sample preparation consists of several subsequent steps

• Each step suffers from loss of material

Workflow simplification

Reproducibility improvement

Low volumes processing (<50uL)

Standardization

Automation

AFA evaluation

for

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Fresh Frozen Tissue project - Covaris collaboration

96 fresh

tissues/cells

96-plex focused ultrasonication

for lysis & protein extraction

96-plex SP3 for

protein clean-up &

digestion*

Label-free LC-MS

Hands-free proteomics

Done in the same plate/no sample transfer

Motivation

• Working in lower

volumes

• Single pot protocol

• Hands-off process

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Fig A : • yields are very linear• Fresh frozen tissue can

be processed directly, in volumes < 100ul

Fig B : • Results are highly

reproducible for a defined quantity of tissue

• Protein group numbersare high

Fig C and D :• More than 84% of the

results have a CV below 0,3%

• Pearson correlations above 0,9

Mol Syst Biol (2020)16:e9111

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From paraffinized tissue to peptides in the same 96-well plate

Motivation

• Further improve protein

extraction for FFPE tissues

(Laser captured

microdissection)

• Make protocol suitable for

automated sample processing

• Include de-paraffinization step

in the same plate

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Allowing low input processing

“The AFA ultrasonication-assisted lysis showed a higher gain in identification of proteins in limited samples.”S. Li et al., 2013

“Focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of 4000 proteins with injection of the equivalent of only 100–200 cells per analysis.”S. Li et al., 2015

Allowing low input processing

26

0

Buffer: 50 mM HEPES, pH 7.5, buffer containing 0.5% Triton X-100 in the presence of EDTA-free complete protease inhibitor cocktail – NO PHENOL

Werth et al., The Plant Journal (2017) 89, 416–426

Three minutes phosphoproteins extraction in C. reinhardtii

“Cell lysis via adaptive focused acoustics is an alternative to traditional sonication methods that reduces protein degradation and aggregation during cell disruption by avoiding sample overheating”

“Importantly, the optimized native extraction provided sufficient material (5.5 mg protein extracted from 0.7 g wet pellet) for subsequent kinomeand phosphopeptide enrichments.”

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Beyond proteins : Metabolites extraction improved by AFA

in S. lividans

“The AFA metabolite extraction is a non-contact technique and compared to the manual vortex/freeze-thaw method both the temperature and cycle times are better controlled thus resulting in more repeatable metabolite extractions and hence more reproducible data. [..] It would [..] be particularly useful for metabolite analyses where high throughput automation is needed.” Kassama et al., 2009

Fig. 4 Relative peak intensities of the significant metabolites between the AFA and manual vortex extraction techniques on three biological strains of Streptomyces lividans (Gram +)

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Cell Lysis: Highly Reproducible Biomolecules Extraction

S. cerevisiae extraction in exponential and stationary growth phasesDifferences in mass spectra observed in exponential and stationary phase

E. coli proteins and metabolites extraction reproducibility

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Selected publications

https://covaris.com/wp-content/uploads/M020103.pdf

Human cells & tissue

Bacteria

Yeast

Plant cells

Recommended input range

Recommended Buffers

S220 E220evolution / E220 ML230 LE220-plus/LE220R-plus

LE220RSc R230

Difficult samples (Yeast/Plants/Bacteria)/high inputs Mammalian cells and tissues-bacteria/small inputs/throughput

Format • Individual glass tube• Individual processing

• Individual glass tube• Glass plates (E220)• Individual processing

• Individual plastic tube – plastic strips

• Parallel processing

• Individual plastic tube – plastic strips and 96 plates

• Parallel processing• Robot compatible

(LE220R-plus)

• Individual plastic tube – plastic strips and 96/384 plates

• Parallel processing

• Robot compatible

• Plastic 96/384 plates• Parallel processing• Robot compatible• On deck integration

Mammalian cells Up to 10^7 TLB Fixed: 50ul – 130µl Fixed: 50ul – 130µl Flexible: 10 to 100µl Flexible: 10 to 100µl Flexible: 10 to 100µl (2 to 20 in 384)

Flexible: 10 to 100µl (2 to 20 in 384)

Mammalian cells Above 10^7 TLB Fixed: 500µl Fixed: 500µl Not recommended Test Test Test

Mammalian Tissue Up to 10mg (above use cryoPREP for

pre-processing)

TLB Fixed: 50ul – 130µl Fixed: 50ul – 130µl Flexible: 75 to 100 µl Flexible: 75 to 100 µl Flexible: 75 to 100 µl TBC

Bacteria Gram +/Gram -

Up to 10^8 TLB Fixed: 50ul – 130µl Fixed: 50ul – 130µl Flexible: 40 to 100µl Flexible: 40 to 100µl Flexible: 40 to 100µl TBC

Bacteria Gram +/Gram -

Above 10^9 TLB Fixed: 500µl Fixed: 500µl Not recommended Not recommended Not recommended Not recommended

Yeast Up to 10^8 TLB Fixed: 50ul – 130µl Fixed: 50ul – 130µl TBC Flexible: 75 to 100 µl Flexible: 75 to 100 µl TBC

Yeast Above 10^9 TLB Fixed: 500µl Fixed: 500µl Not recommended Not recommended Not recommended Not recommended

Plants/Algae/Fungi Depends on starting material – usually pre-processed on cryoPREP

TLB Fixed: 1mL and above Fixed: 1mL and above Not recommended Not recommended Not recommended Not recommended

Other: biofluids (plasma, saliva, CSF,

tears...)

N/A Saliva Saliva Plasma - WIP Plasma - WIP Plasma - WIP TBC

Other : enzymatic digestion (trypsin,

Lys C,...)

N/A Published Published WIP WIP WIP WIP

Proteomics Instrument Decision Chart

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What AFA (adaptive focused acoustics) bringsto your sample prep

• Extraction : Controlled, Isothermal, No contact Energy

• All sample types

• You can use your buffer

• You can choose any clean-up solution

• Works in broad volume range (<20ul to >10mL)

• Hands off

• Compatible with single tube and up to 96 format processing and beyond

Sameprecision in individual

tubes or plates

High Reproducibility

Lesssteps

Single pot

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What AFA (adaptive focused acoustics) bringsto your sample prep

• Extraction : Controlled, Isothermal, No contact Energy

• All sample types

• You can use your buffer

• You can choose any clean-up solution

• Works in broad volume range (<20ul to >10mL)

• Hands off

• Compatible with single tube and up to 96 format processing and beyond

Sameprecision in individual

tubes or plates

High Reproducibility

Lesssteps

Single pot

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For more information, please contact:

Nicolas Autret, Ph.D.

Business Development Manager Europe – Proteomics Solutions

nautret@covaris.com

THANK YOU!

Covaris LtdUnit 3, Brighton Office CampusHunns Mere WayWoodingdean BrightonBN2 6AHUnited KingdomTel: +44 (0)845 872 0100Fax: +44 (0) 845 384 9160EUCustomerService@covaris.com

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Covaris offers a range of buffers developed for :

• LC-MS

• MALDI

• ELISA, Multiplex

• Western Blot

• 2D PAGE

Ready-to-use Buffers for Optimized Sample Prep

Skea et al., 2012

Buffers description:

truXTRAC Protein Extraction Buffer N•Physiological Tris-Based buffer for extraction of proteins in their native conformation•Extracted proteins are directly compatible with affinity enrichment, enzyme activity and protein assays•Extraction with AFA provides high yield of native proteins

truXTRAC Protein Extraction Buffer SuperB•Optimized for native applications preserving protein conformation and biological activity.•Improved protein yields while preserving native conformation, antigenicity, and biological activity

truXTRAC Protein Extraction Buffer DF•Efficient detergent-free protein extraction•Urea-based reagent includes optional additives EDTA, protease inhibitors and NaCl to allow optimization

truXTRAC Protein Extraction Buffer TP•Maximizes total protein yields from cells and tissues with an optimized urea, thiourea, and zwitterionic detergent•Provides a stable dry chemical blend and includes an ion exchange resin to yield higher purity of reagents•Low conductivity of freshly prepared reagents makes it ideal for applications such as IEF and 2D gel

NEW! truXTRAC Protein Extraction Buffer TLB• SDS based buffer• Includes a reagent to optimize reverse

crosslinking for FFPE extraction

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Ready-to-use Buffers : FAQs

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Human cells & tissue

Bacteria

Yeast

Plant cells

Proteins

DNA RNA

Chromatin

Lyse Extract Process

Small Molecules

Lipids

Ligation

Hybridization

Nanoemulsion

Shearing

Protein processing

• Low to high volumes (10ul <-> 30ml)

• Low to high content (few cells <->

tissues)

• 1 to 384 samples at a time

• Broad spectrum of extraction

protocols (cells, tissue, solutions)

• Gold standard technique for DNA shearing in NGS

• Increasing number of FFPEapplications

Countless possibilities

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Insert Plate into

LE220R-plus

Robotic Arm Moves Plate from Service

Position

oneTUBE Compatible with any Liquid Handler

LE220R-plus Full Functionality Using Robotic Arms

Integrate with any robotic arm to a liquid handler, plate hotel, or any other robotic system

37

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Photos courtesy of ARUP Laboratories

24/7, 365 Facility Operation

Success Story

• Enhanced sample traceability

• Reduced human intervention

• Increased throughput

• Walk-away solution

• Highest reproducibility

• Customized DNA fragmentation Covaris LE220R-plus

• Tunable for various sample types and specimen quality

• Robotic loading with curtain to maintain safety standard

Fully Automatable Solutions

ARUP Laboratories with Covaris

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Small footprint on a liquid handler

Revolutionize your applications with AFA on-deck

Non-Stop workflow (cell lysis -> extraction -> shearing…)

• High-throughput processing

• Multi-applications

• Compatible with most liquid handlers

• Optimal footprint

• Designed for fully automated error-proof and traceable workflows

• AFA tubes 8 strip 96 wells plates for optimized performance in standard format

• Enable one-pot process, cost-efficiency, and fast turnaround-time (TAT)

AFA-tubes in SBS formatVersatile miniaturized Focused-ultrasonicator

The Future is Already Here

R230: Re-think Sample Prep Automation

Small footprint on a liquid handler