Cell LysisA presentation of AFA-energeticsreg enabled Applications for high resolution sample preparation

Covaris

Table of Contents

Introduction 3

AFABenefits 3

High-throughputClinicalProteomicsfromCellsFreshFrozenTissueandFFPESamples 5

PreservingProteinIntegrityExtractionofNativeProteins 6

LowInputExtraction 8

Hard-to-lyseSamples 9

CellLysisinEukaryotes 9

CellLysisofPatientDerivedXenografts(PDXs) 10

CellLysisinProkaryotes 10

VersatilityofAFA 11

3

IntroductionThechoiceofsample-preparationmethodisacriticalfirststepinproteomicandmetabolomicstudiesbecauseitisanessential

partofchromatographicandspectroscopicanalysesItaffectsboththeobservedmoleculecontentandthedownstreambiological

interpretation

Anidealsample-preparationmethodshould

bull Beasnon-selectiveaspossible

bull Preventlossandordegradationduringthepreparationprocedure

bull Avoidcontamination

bull Enablehigh-throughputprocessing

bull Ensurereproducibility

bull Becompatiblewiththedownstreamanalyticalmethod

TheimportanceoftheseaspectsemergedinthelastdecadewithmoresophisticatedtechniqueslikeFASP(FilterAssistedSample

Preparation)andtheuseofhighgradereagentsinordertoreducethepossibleinterferencewiththeintendedanalysistechniques

likeLiquidChromatography-MassSpectrometry(LC-MS)Inmostcasessamplepreparationremainsinconsistentandtimeconsuming

CovarisAdaptiveFocusedAcousticsreg(AFAreg)cansubstantiallyreducesamplevariabilityandoverallturn-around-timetosimplifythe

workflowforMS

AFA Benefits

Inadditiontosamplepreparationimprovementsthereisadesiretoreducethesamplesizeandincreasethethroughputwhile

maintainingorachievinghigherreproducibilityevenwhenworkingwithdifficulttoprocesssamples(plantsyeasthardmammalian

tissuesFFPEblocks)

Ahugevarietyofworkflowsexisttoobtainthehighestyieldandpuritydependentonthespeciesorganismsthesampletypeandthe

targetedmoleculesBiomoleculediversitypresentsanotherchallengeforexampleproteinswithposttranslationalmodificationcanbe

veryfragileormembraneproteinsveryinsolubleThiscanleadtogreatvariabilityinrecoveryandqualityespeciallyincorelabswhich

requireastandardizedworkflowsuitableforawiderangeofbiologicalsamples

ThisdocumenthighlightstherecentdevelopmentsofAdaptiveFocusedAcousticsforisolatingproteinsandotherbiomarkersfrom

difficult-to-treatsamplesforavarietyofinputsincludingcomplextissuetosinglecellanalysisforhightotalproteinyieldsaswellas

nativeproteinspreservationfocusedultrasoundsensureareproduciblenon-contactandisothermaltreatmentofeachsampleleading

tohigherqualityextractionandbiomarkerpreservation

AnalysisSampleCollection Cell Lysis ProcessingExtraction

bull Standardizesamplepreparation

bull Preservesampleheterogeneity

bull High-throughputautomationready

bull Reproducible

bull Compatiblewithdownstreamanalysismethods

bull Versatilemostsampletypes

bull Streamlineworkflow

4

References bull ProteomicChallengesSamplePreparationTechniquesforMicrogram-QuantityProteinAnalysisfromBiologicalSamplesPFeist

etalIntJMolSci2015163537-3563DOI103390ijms16023537

bull ChallengesinbiomarkerdiscoverywithMALDI-TOFMSJHajduketalClinicaChimicaActaVolume4581July2016Pages

84-98DOI101016jcca201604033

bull IntegralmembraneproteinsinproteomicsHowtobreakopentheblackboxOVitetalJournalofProteomics153(2017)

8ndash20DOI101016jjprot201608006

bull ModernProteomicsndashSamplePreparationAnalysisandPracticalApplicationsAdvancesinExperimentalMedicineandBiology-

pp23-62ndash2017DOI101007978-3-319-41448-5_4

bull SelectingSamplePreparationWorkflowsforMassSpectrometry-BasedProteomicandPhosphoproteomicAnalysisofPatient

SampleswithAcuteMyeloidLeukemiaHernandez-VallaresetalProteomes2016424DOI103390proteomes4030024

Table of Contents

Introduction 3

AFABenefits 3

High-throughputClinicalProteomicsfromCellsFreshFrozenTissueandFFPESamples 5

PreservingProteinIntegrityExtractionofNativeProteins 6

LowInputExtraction 8

Hard-to-lyseSamples 9

CellLysisinEukaryotes 9

CellLysisofPatientDerivedXenografts(PDXs) 10

CellLysisinProkaryotes 10

VersatilityofAFA 11

3

IntroductionThechoiceofsample-preparationmethodisacriticalfirststepinproteomicandmetabolomicstudiesbecauseitisanessential

partofchromatographicandspectroscopicanalysesItaffectsboththeobservedmoleculecontentandthedownstreambiological

interpretation

Anidealsample-preparationmethodshould

bull Beasnon-selectiveaspossible

bull Preventlossandordegradationduringthepreparationprocedure

bull Avoidcontamination

bull Enablehigh-throughputprocessing

bull Ensurereproducibility

bull Becompatiblewiththedownstreamanalyticalmethod

TheimportanceoftheseaspectsemergedinthelastdecadewithmoresophisticatedtechniqueslikeFASP(FilterAssistedSample

Preparation)andtheuseofhighgradereagentsinordertoreducethepossibleinterferencewiththeintendedanalysistechniques

likeLiquidChromatography-MassSpectrometry(LC-MS)Inmostcasessamplepreparationremainsinconsistentandtimeconsuming

CovarisAdaptiveFocusedAcousticsreg(AFAreg)cansubstantiallyreducesamplevariabilityandoverallturn-around-timetosimplifythe

workflowforMS

AFA Benefits

Inadditiontosamplepreparationimprovementsthereisadesiretoreducethesamplesizeandincreasethethroughputwhile

maintainingorachievinghigherreproducibilityevenwhenworkingwithdifficulttoprocesssamples(plantsyeasthardmammalian

tissuesFFPEblocks)

Ahugevarietyofworkflowsexisttoobtainthehighestyieldandpuritydependentonthespeciesorganismsthesampletypeandthe

targetedmoleculesBiomoleculediversitypresentsanotherchallengeforexampleproteinswithposttranslationalmodificationcanbe

veryfragileormembraneproteinsveryinsolubleThiscanleadtogreatvariabilityinrecoveryandqualityespeciallyincorelabswhich

requireastandardizedworkflowsuitableforawiderangeofbiologicalsamples

ThisdocumenthighlightstherecentdevelopmentsofAdaptiveFocusedAcousticsforisolatingproteinsandotherbiomarkersfrom

difficult-to-treatsamplesforavarietyofinputsincludingcomplextissuetosinglecellanalysisforhightotalproteinyieldsaswellas

nativeproteinspreservationfocusedultrasoundsensureareproduciblenon-contactandisothermaltreatmentofeachsampleleading

tohigherqualityextractionandbiomarkerpreservation

AnalysisSampleCollection Cell Lysis ProcessingExtraction

bull Standardizesamplepreparation

bull Preservesampleheterogeneity

bull High-throughputautomationready

bull Reproducible

bull Compatiblewithdownstreamanalysismethods

bull Versatilemostsampletypes

bull Streamlineworkflow

4

References bull ProteomicChallengesSamplePreparationTechniquesforMicrogram-QuantityProteinAnalysisfromBiologicalSamplesPFeist

etalIntJMolSci2015163537-3563DOI103390ijms16023537

bull ChallengesinbiomarkerdiscoverywithMALDI-TOFMSJHajduketalClinicaChimicaActaVolume4581July2016Pages

84-98DOI101016jcca201604033

bull IntegralmembraneproteinsinproteomicsHowtobreakopentheblackboxOVitetalJournalofProteomics153(2017)

8ndash20DOI101016jjprot201608006

bull ModernProteomicsndashSamplePreparationAnalysisandPracticalApplicationsAdvancesinExperimentalMedicineandBiology-

pp23-62ndash2017DOI101007978-3-319-41448-5_4

bull SelectingSamplePreparationWorkflowsforMassSpectrometry-BasedProteomicandPhosphoproteomicAnalysisofPatient

SampleswithAcuteMyeloidLeukemiaHernandez-VallaresetalProteomes2016424DOI103390proteomes4030024

3

IntroductionThechoiceofsample-preparationmethodisacriticalfirststepinproteomicandmetabolomicstudiesbecauseitisanessential

partofchromatographicandspectroscopicanalysesItaffectsboththeobservedmoleculecontentandthedownstreambiological

interpretation

Anidealsample-preparationmethodshould

bull Beasnon-selectiveaspossible

bull Preventlossandordegradationduringthepreparationprocedure

bull Avoidcontamination

bull Enablehigh-throughputprocessing

bull Ensurereproducibility

bull Becompatiblewiththedownstreamanalyticalmethod

TheimportanceoftheseaspectsemergedinthelastdecadewithmoresophisticatedtechniqueslikeFASP(FilterAssistedSample

Preparation)andtheuseofhighgradereagentsinordertoreducethepossibleinterferencewiththeintendedanalysistechniques

likeLiquidChromatography-MassSpectrometry(LC-MS)Inmostcasessamplepreparationremainsinconsistentandtimeconsuming

CovarisAdaptiveFocusedAcousticsreg(AFAreg)cansubstantiallyreducesamplevariabilityandoverallturn-around-timetosimplifythe

workflowforMS

AFA Benefits

Inadditiontosamplepreparationimprovementsthereisadesiretoreducethesamplesizeandincreasethethroughputwhile

maintainingorachievinghigherreproducibilityevenwhenworkingwithdifficulttoprocesssamples(plantsyeasthardmammalian

tissuesFFPEblocks)

Ahugevarietyofworkflowsexisttoobtainthehighestyieldandpuritydependentonthespeciesorganismsthesampletypeandthe

targetedmoleculesBiomoleculediversitypresentsanotherchallengeforexampleproteinswithposttranslationalmodificationcanbe

veryfragileormembraneproteinsveryinsolubleThiscanleadtogreatvariabilityinrecoveryandqualityespeciallyincorelabswhich

requireastandardizedworkflowsuitableforawiderangeofbiologicalsamples

ThisdocumenthighlightstherecentdevelopmentsofAdaptiveFocusedAcousticsforisolatingproteinsandotherbiomarkersfrom

difficult-to-treatsamplesforavarietyofinputsincludingcomplextissuetosinglecellanalysisforhightotalproteinyieldsaswellas

nativeproteinspreservationfocusedultrasoundsensureareproduciblenon-contactandisothermaltreatmentofeachsampleleading

tohigherqualityextractionandbiomarkerpreservation

AnalysisSampleCollection Cell Lysis ProcessingExtraction

bull Standardizesamplepreparation

bull Preservesampleheterogeneity

bull High-throughputautomationready

bull Reproducible

bull Compatiblewithdownstreamanalysismethods

bull Versatilemostsampletypes

bull Streamlineworkflow

4

References bull ProteomicChallengesSamplePreparationTechniquesforMicrogram-QuantityProteinAnalysisfromBiologicalSamplesPFeist

etalIntJMolSci2015163537-3563DOI103390ijms16023537

bull ChallengesinbiomarkerdiscoverywithMALDI-TOFMSJHajduketalClinicaChimicaActaVolume4581July2016Pages

84-98DOI101016jcca201604033

bull IntegralmembraneproteinsinproteomicsHowtobreakopentheblackboxOVitetalJournalofProteomics153(2017)

8ndash20DOI101016jjprot201608006

bull ModernProteomicsndashSamplePreparationAnalysisandPracticalApplicationsAdvancesinExperimentalMedicineandBiology-

pp23-62ndash2017DOI101007978-3-319-41448-5_4

bull SelectingSamplePreparationWorkflowsforMassSpectrometry-BasedProteomicandPhosphoproteomicAnalysisofPatient

SampleswithAcuteMyeloidLeukemiaHernandez-VallaresetalProteomes2016424DOI103390proteomes4030024

4

References bull ProteomicChallengesSamplePreparationTechniquesforMicrogram-QuantityProteinAnalysisfromBiologicalSamplesPFeist

etalIntJMolSci2015163537-3563DOI103390ijms16023537

bull ChallengesinbiomarkerdiscoverywithMALDI-TOFMSJHajduketalClinicaChimicaActaVolume4581July2016Pages

84-98DOI101016jcca201604033

bull IntegralmembraneproteinsinproteomicsHowtobreakopentheblackboxOVitetalJournalofProteomics153(2017)

8ndash20DOI101016jjprot201608006

bull ModernProteomicsndashSamplePreparationAnalysisandPracticalApplicationsAdvancesinExperimentalMedicineandBiology-

pp23-62ndash2017DOI101007978-3-319-41448-5_4

bull SelectingSamplePreparationWorkflowsforMassSpectrometry-BasedProteomicandPhosphoproteomicAnalysisofPatient

SampleswithAcuteMyeloidLeukemiaHernandez-VallaresetalProteomes2016424DOI103390proteomes4030024

5

High-throughput Clinical Proteomics from Cells Fresh Frozen Tissue and FFPE SamplesHigh-throughputandstreamlinedworkflowsareessentialinclinicalproteomicsforrobustreliableandcomprehensiveproteome

profilingResearchersarelookingforstandardizedprotocolstoprocessvarioussamplesincludingfresh-frozentissueFFPEtissueor

bloodThetwopapersbelowexemplifyhowCovarisAFAcansetnewstandardsinsamplepreparationforproteomics

ClinicalpracticerequiresreducedhumaninterventionandtheabilitytoprocesssmallinputswithsufficientthroughputHowever

proteinextractionisstilllargelyamanualprocesswithmanystepsincludinglysisandhomogenizationofthesampleforproperprotein

solubilizationandstabilizationAFA-energeticssimplifiestheworkflowandharmonizesprotocolwhileenablingfullautomation

includingintegrationlaboratoryroboticsoftheprocess

AutomatedsamplepreparationwithSP3forlow-inputclinicalproteomics

TMuellerJKrijgsveldetalMolecularSystemsBiology16e9111|2020ndashDOI1015252msb20199111

ThispaperisthefirstpublishedautomatedmethodforproteinsamplepreparationusingCovarisTheauthorsdemonstrated1)the

abilitytoworkwithlowvolumes2)thepossibilitytoworkonbothcellsandtissuesand3)theefficiencyofasinglepotworkflowto

extractandidentifyproteinsreproduciblyandconsistently

InclinicalproteomicsFFPEblocksrepresentoneofthelargestsourcesofarchivedsamplesTraditionallyduetoinefficientor

incompletedeparaffinizationanddecrosslinkingFFPEanalysishassufferedfrompoorproteinrecoverylackofreproducibilityandlack

ofspeedTheuniquecombinationofCovarisAFAandProtiFitradeS-Trapstradeallowsforarapidstreamlinedapproachusingonetubeand

onecolumnThisnovelworkflowaffordsthehighestyieldsofproteinsnumberofidentificationsandthemostreproducibleFFPE

sampleprocessingInadditionitiswellsuitedforhigh-throughputworkflows

Keywords FFPE oncology cancer research paraffin

References bull HYPERsolHigh-QualityDatafromArchivalFFPETissueforClinicalProteomicsDMMarchioneetal2020

DOI101021acsjproteome9b00686JProtRes2020192973-983

- TheresultspresentedinthisarticleindicatethesuperiorityofcombiningAFASDSbasedbufferS-Trapcolumns(describedasHYPERSOL)

overtraditionalmethodstoefficientlyextractproteinsfromdifferentFFPEsamplesincludingoldsamplesstoredformorethan17years

bull USHUPO2019posterTotalSolubilizationofFFPEsamplesforHighThroughputClinicalProteomicsJWilsonJWojciketal

httpsabrf2019gorgesappsusnode3876

- ThisworkisthefoundationoftheHYPER-solpaper

bull HUPO2018posterUniversalSampleProcessingofMultipleSampleTypesForReproducibleProteomicSamplePreparation

JWilsonVMeyyappanetalhttpscovariscomwp-contentuploadsHUPO-2018-ProtiFi-Covaris-Posterpdf

- ThisposterpresentsauniversalprotocolforproteinextractionondifficultsampleslikeFFPEbrainorpancreasDatashowshowefficient

thisprotocolistoisolateproteinsthatcanbemissedbyothermethods

6

Preserving Protein Integrity Extraction of Native ProteinsWhenconsideringextractionitisimportanttodefinewhatpopulationofproteinsisofinterestasitisnearlyimpossibletofind

conditionsthatwillaccommodateallclassesofproteinswithcomparableefficiencyHerewefocusonscientificpublications

communicationsdescribingmethodsthatmaintainthenativestateoftheproteinsThiswillallowthestudyoftheirposttranslational

modifications(PTMs)likephosphorylationorubiquitinationormorecomplexdownstreamapplicationssuchasactivityassaysas

examples

Keywords post translational modifications (PTM) native protein phosphoprotein ubiquitination glycosylation

References bull Robustpre-analyticalsamplepreparationprocesspreservestheaccuracyandfidelityofproteinphosphorylationstatesSmejkal

etalHUPO2012-poster

- ThispostershowstheefficiencyofAFAtodeliveroverdouncehomogenizationwithregardstoproteinqualityandquantity

bull Combinedphospho-andglycoproteomeenrichmentinnephrocalcinosistissuesofphytate-fedratsTTranetalRapidCommun

MassSpectrom2013272767ndash2776DOI101002rcm6742

- ThispaperstressestheimportanceofpreservingproteinsintegrityduringsamplepreparationinparticularwhenstudyingPTMslike

phosphorylationandglycosylation

bull Comprehensiveandsensitiveproteogenomicsdataanalysisstrategybasedoncomplementarymulti-stagedatabasesearch

IHMadaretalInternationalJournalofMassSpectrometryVolume427April2018Pages11-19DOIdxdoiorg101016j

ijms201708015

- Sensitivitywaslookedafterinthisproteogenomicspaperstudyingtheproteomeofhumancancertissues

bull Ahigh-efficiencycellularextractionsystemforbiologicalproteomicsDhabariaetalJofProteomeRes2015August714(8)

3403ndash3408DOI101021acsjproteome5b00547

- InthispapertheyarelookingtomaximizetheextractionofcellularproteinswhileminimizingtheirdenaturationAFAcombinedwithan

optimizeddetergentsystempermittedefficientnativeproteomeextraction

bull Useoffocusedultrasonicationinactivity-basedprofilingofdeubiquitinatingenzymesintissueNandurietalAnalBiochem

2016December155159ndash13DOI101016jab201609016

- ThispapershowscomparisonofvarioussampleprepmethodsAFAgivesthebestresultsforfollow-upofubiquitination

bull Mappingproteinsignalpathwayinteractioninsarcomabonemetastasislinkagebetweenrankmetalloproteinasesturnoverand

growthfactorsignalingpathwaysContietalClinExpMetastasis2014Jan31(1)15-24DOI101007s10585-013-9605-6

- AFAcombinedwithcryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedbyRPPAtechnique

bull Integratedanalysisofglobalproteomephosphoproteomeandglycoproteomeenablescomplementaryinterpretationofdisease-

relatedproteinnetworksJMParketal2015ScientificReports|518189DOI101038srep18189

- Reproducibleandefficientnativeproteinextractionwaskeyinthislarge-scaleproteomeanalysisofthreegastriccancerpatients

integratingphospho-andglycoproteinswherebothcryoPREPandAFAwereused

bull Optimizedcross-linkingmassspectrometryforin situinteractionproteomicsZSeretal2018BioRxivDOI101101393892

- AFAwasusedtofavourextractionofnativecomplexeswhilestudyingprotein-proteininteractionsusingcross-linkingmassspectrometry

(XL-MS)

bull MappingProteinSignalPathwayInteractioninSarcomaBoneMetastasisLinkageBetweenRankMetalloproteinasesTurnover

andGrowthFactorSignalingPathwaysContietalClinExpMetastasis2014Jan31(1)15-24

DOI101007s10585-013-9605-6

- AFAcombinedwiththecryoPREPallowedforefficientextractionandpreservationofsignalingproteinsfurtheranalyzedby

RPPAtechnique

7

bull 6-PhosphogluconateDehydrogenaseLinksCytosolicCarbohydrateMetabolismtoProteinSecretionviaModulationof

GlutathioneLevelsHLietal2019-CellChemicalBiology261306ndash1314ndashDOI101016jchembiol201905006

- ReproduciblecelllysiswasperformedoncellpelletsusingAFAforLC-MSanalysis

bull Highsensitivityquantitativeproteomicsusingautomatedmultidimensionalnanoflowchromatographyandaccumulatedion

monitoringonquadrupole-OrbitraplineariontrapmassspectrometerPCifanietalMolCellProteomics2017

Nov16(11)2006-2016DOI101074mcpRA117000023

- AuthorssoughttoincreasesensitivityofdetectionincludingmodifiedproteinsImprovedsamplepreparationwasoneof

thepre-requisites

bull ProbingtheglobalkinomeandphosphoproteomeinChlamydomonasreinhardtiiviasequentialenrichmentandquantitative

proteomicsEWerthetalThePlantJournal(2017)89416ndash426DOI101111tpj13384

- TheauthorswerelookingforamethodbeingeffectivefordisruptingChlamydomonascellsandimprovenativeproteinextractionThey

hadtheobjectiveofmaximizingyieldtoaccommodatetherequirementforhighamountsofproteininthekinomeandphosphoproteome

enrichmentstepsuseddownstream

bull ThephosphorylatedredoxproteomeofChlamydomonas reinhardtiiRevealingnovelmeansforregulationofproteinstructure

andfunctionMcConnelletalRedoxBiologyVolume17July2018Pages35-46DOIdoiorg101016jredox201804003

- TheHickslab(seeWerthetal)describesdemonstrationofprotein-levelenrichmentwithAFAofreversiblyoxidizedproteoformsin

Chlamydomonasreinhardtiiwithsubsequentphosphopeptideanalysistodeterminetheextentofphosphorylationintheredoxthiol

proteome

bull InvestigatingtheeffectoftargetofrapamycinkinaseinhibitionontheChlamydomonasreinhardtiiphosphoproteomefrom

knownhomologstonewtargetsEwerthetalNewPhytologist(2018)221247ndash260DOI101111nph15339

- UsingAFAforextractingphosphoproteinsWertetalachievedextensivecoverageoftheTOR-modulatedphosphoproteomein

Chlamydomonasusingaquantitativelabel-freeapproach

bull MassSpectrometryndashBasedProteomicsRevealsPotentialRolesofNEK9andMAP2K4inResistancetoPI3KInhibitioninTriple-

NegativeBreastCancersMundtetalCancerRes2018May1578(10)2732-2746DOI1011580008-5472

- AnotherpaperontheuseofAFAforPDXs(seepapersfromWangandNtai)centeredonphosphoproteogenomicstounderstand

resistancemechanismsinbreastcancer

8

Low Input ExtractionRecentlymoreandmorestudieshavebeenconductedonlownumberofcellslt10000Theabilitytoreachtheindividualcelllevel

canyieldessentialdetailstodistinguishbetweencelltypesanddeciphertheirsignalingactivitiesItisalsoarequirementtobeable

toworkwithhigh-throughputsThoselowinputsamplesmustbeprocessedinsmallvolumes10to200microLorlesstomaintaina

sufficientconcentrationwhileminimizingthelossbetweeneachstepoftheworkflowAnotherconstraintisensuringthateverytube

willbetreatedidenticallyandifpossiblesimultaneouslyorwithinashorttimeframeToensuretheseparametersaremetresearchers

havedevelopedhigh-throughputprotocolsusing96wellplatesFurthermoreincertainprotocolsthecombinationofstepsinso

calledldquoonepotrdquoreactionsreducedthecomplexityoftheworkflowsandallowsforbetterstandardization

Keywords low cell extraction low input cell lysis single cell

References bull AnIntegratedPlatformforIsolationProcessingandMassSpectrometry-basedProteomicProfilingofRareCellsinWholeBlood

SLietalMolecularampCellularProteomics141672ndash16832015DOI101074mcpM114045724

- Withcontrolledextractionparameterstheauthorsachievedzeptomoledetectionsensitivityresultinginidentificationof4000proteins

fromtheequivalentof100to200cells

bull Mass-spectrometryofsinglemammaliancellsquantifiesproteomeheterogeneityduringcelldifferentiationBBudniketal

Genome Biology201819161DOI101186s13059-018-1547-5

- AFAwasusedtoensureminimallossofproteinsandobviatechemicalsthatmayunderminepeptideseparationandionizationorsample

cleanupthatmayincursignificantlosses

bull Integratedmicroscaleanalysissystemfortargetedliquidchromatographymassspectrometryproteomicsonlimitedamountsof

enrichedcellpopulationsJGMartinetalAnalChem2013Nov1985(22)10680-5DOIdxdoiorg101021ac401937c

- ThispaperisshowingAFAuseinacontextoflowcelllowinputextraction(lt5000cells)

bull LymphaticexosomespromotedendriticcellmigrationalongguidancecuesMBrownetalJCellBiol2018Jun4217(6)2205-

2221DOI101083jcb201612051

- Gentleextractionwithproteinconservationledtotheidentificationofgt1700proteinsinexosome-richendothelialvesicles(EEVs)to

understandwhatdrivesthereleaseofEEVsbylymphaticendothelialcells

bull HighSensitivityMicroproteomicAnalysisofRareSamplesbyPorousLayerOpenTubular(PLOT)ColumnsCoupledwithMass

SpectrometrySLietalposterndashASMS2013

- AnotherexampleshowingtheupsidesofusingAFAwhenworkingwithlownumberofcellscomparedtoothertraditionalextraction

techniques

9

Hard-to-lyse SamplesSamplepreparationisalwaysaboutoptimizationthereisasignificantnumberofparametersthatcanaffecttheefficiencyofbiomarker

recoverySomeorganismshaveveryrigidmembraneconstituentswhileotherscanhaveacellwallontopoftheirmembraneand

theinsolubilityofsomecomponentscandrasticallydecreasethequantityofdesiredbiomoleculesAFAhasshowntobeefficientin

processingawidevarietyofstartingmaterialsincludingplantsbacteriayeastorhardmammaliantissuelikemuscle

ReferencesCell Lysis in Eukaryotes bull DihydrolipoyldehydrogenaseasapotentialUVBtargetinskinepidermisusinganintegratedapproachoflabel-freequantitative

proteomicsandtargetedmetaboliteanalysisMoonetalJournalofProteomicsVolume11718March2015Pages70-85

DOIdxdoiorg101016jjprot201412016

- AFAwasusedtodisruptdifficult-to-lyseskinsampleswhileensuringgoodrecoveryofproteinsandmetabolites

bull High-ThroughputSimultaneousAnalysisofRNAProteinandLipidBiomarkersinHeterogeneousTissueSamplesReiseretal

ClinicalChemistry57111545-15552011DOI101373clinchem2010157743

- Theauthorsefficientlyextractedseveraltypesofbiomarkersfromdifficulttissue(atheroscleroticplaqueandtumortissue)usingcryoPREP

fortissuepulverizationandAFAmethodforsuccessfulproteinextraction

bull ArapidstandardizedproteinextractionmethodusingadaptivefocusedacousticsforidentificationofmycobacteriabyMALDI-

ToFMSLTAdamsetalDiagnosticMicrobiologyandInfectiousDisease86(2016)284ndash288

DOI101016jdiagmicrobio201606001

- ThispaperevaluatesAFAtorapidlyextractmycobacterialpeptidesandalsoforitsabilitytoinactivatequicklyallspeciesofmycobacteria

bull PlasmamembraneproteomeinArabidopsisandriceSKomatsuProteomics200884137ndash4145DOI101002

pmic200800088

- Areviewhighlightingtheadvantagesofacoustictechniquestohomogenizeproteinpelletsfromvariousplanttissues

bull AMicroscaleYeastCellDisruptionTechniqueforIntegratedProcessDevelopmentStrategiesMDWengeretalBiotechnol

Prog200824606minus614DOI101021bp070359s

- InthisyeaststudyAFAnon-contactapproachwaskeytolyseefficientlyhighquantitiesofcellsdespiteaveryrigidcellwall

bull PeptidomicsanalysisoftransientregenerationintheneonatalmouseheartYFanetalJCellBiochem2017Sep118(9)2828-

2840DOI101002jcb25933

- UseofAFAforpeptidomics(thebridgebetweenproteomeandmetabolome)onmousehearttissue

bull Developmentofahigh-throughputmicroscalecelldisruptionplatformforPichia pastorisinrapidbioprocessdesignBlahaetal

BiotechnolProg2018Jan34(1)130-140DOI101002btpr255

- Objectivewastodevelopanautomatedminiaturizedhigh-throughputnon-contactscalableplatformbasedonAdaptiveFocused

Acoustics(AFA)todisruptP pastorisandrecoverintracellularheterologousproteinConclusionshowsthatAFAcanbeusedvery

efficientlyinawiderangeofapplications

bull AcousticTechnologyforHigh-PerformanceDisruptionandExtractionofPlantProteinsMToorchietalJournalofProteome

Research200873035ndash3041DOI101021pr800012c

- AuthorsdescribehowAFAperformsfarbetteronplantsamplesthanwaterbathsonicationbyproducinghigh-quality2Dgelsand

minimizingtheprocessingtimerequiredforhigh-throughputproteomicsresearch

bull SoybeanProteomicsforUnravelingAbioticStressResponseMechanismZHossainetalJProteomeRes201312114670-

4684DOI101021pr400604b

- AnalyzingdifferentpreparationmethodstheauthorsdescribeCovarisprocessingasresultingldquoInaclearerproteinpatternthantheother

conventionalmethodsrdquo

10

Cell Lysis of Patient Derived Xenografts (PDXs)AFAisveryefficientforxenograftsAlongwiththepaperfromMundtetalTostudyphosphoproteinsotherteamshaveuseditfor

thispurpose

bull BreasttumorseducatetheproteomeofstromaltissueinanindividualizedbutcoordinatedmannerXWangetalSciSignal

2017Aug810(491)DOI101126scisignalaam8065

- Studyingheterogeneitybetweentumorsrequiresahighdegreeofsensitivityandgoodqualityproteinextractionasshownhereonbreast

xenografts

bull IntegratedBottom-UpandTop-DownProteomicsofPatient-DerivedBreastTumorXenograftsINtaiMolecularampCellular

Proteomics15101074DOI101074mcpM114047480

- Authorsdescribethefirstlarge-scaleintegrationofgenomicbottom-upandtop-downproteomicmeasuringdifferentialexpressionof

proteinsandproteoforms

Cell Lysis in Prokaryotes bull TheRoleofCadaverineSynthesisonPneumococcalCapsuleandProteinExpressionMFNakamyaetalMedSci(Basel)2018

Jan196(1)DOI103390medsci6010008

- UseofAFAtodisruptS pneumoniaecapsule

bull UseofFocusedAcousticsforCellDisruptiontoProvideUltraScale-DownInsightsofMicrobialHomogenizationandits

BioprocessImpactmdashRecoveryofAntibodyFragmentsfromrecE coliQLietalBiotechnologyandBioengineeringVol109

No8August2012DOI101002bit24484

- ThisstudydemonstratessuperiorefficiencyofAFAoverclassicalsonication

bull Anultrascaled-downapproachtostudytheinteractionoffermentationhomogenizationandcentrifugationforantibody

fragmentrecoveryfromrecE coliQLietalBiotechnologyandBioengineering2013Aug110(8)2150-60

DOI101002bit24891

- InthisstudyauthorsapplyAFA(definedastheirmethodofchoiceintheupperpaper)toE coliforhomogenizationanddisruptionpurpose

inthecontextofultrascaleddownoptimization

bull AssessmentoftheManufacturabilityofEscherichiacoliHighCellDensityFermentationsMAPerez-PardoetalBiotechnol

Prog271488ndash14962011DOI101002btpr644

- AFAhelpedinassessingthebestphysiologicalandbiologicalconditionsforfermentationstartingfromultrascaleddownquantities

11

Versatility of AFACovarisAFAhasdemonstrateditsefficiencytodisruptcellsofgreatdiversityandformanydifferentobjectivesintherecoveryof

intracellularbiomoleculesincludingmetabolitesantibodyfragmentsproteinsandproteinsubunitsmembraneproteinsandlipids

AllofthesehavebeenisolatedwithhighefficiencyandexcellentpreservationwithAFAThisprovedtobeofparticularinterestfor

proteogenomicsstudiesAFAalsoprovidesvaluableadvantagesascomparedtootherapplicationsasitcanenhancethespeedand

qualityoftrypticdigestionandforhydrogelssolubilization

Keywords high throughput label free trypsin digestion stem cells western blotting proteogenomics cross linked MS (XL-MS)

References bull OptimizedCross-linkingMassSpectrometryforInSituInteractionProteomicsZSerAKentsisetalJProteomeRes201918

62545-2558DOI101021acsjproteome9b00085

- Crosslinkingmassspectrometry(XL-MS)requiresoptimalmethodsfortheisolationofcross-linkedpeptidesfromproteincomplexesincluding

properproteinextractionandpreservationasexemplifiedbyAFA

bull ProteomeGeneratorAFrameworkforComprehensiveProteomicsBasedondeNovoTranscriptomeAssemblyandHigh-

AccuracyPeptideMassSpectralMatchingZifanietalJProteomeRes201817113681-3692

DOI101021acsjproteome8b00295

- CovarisAFA-assistedextractionisusedforgenome-scaleandquantitativemeasurementsofbiologicalproteomes(proteogenomics)as

allowedbymodernmassspectrometry

bull PGBD5promotessite-specificoncogenicmutationsinhumantumorsAGHenssenetalNatureGeNeticsemspVOLUME49|

NUMBER7|JULY2017DOI101038ng3866

- StudyinggenomicrearrangementsofPGBD5whichtheywereabletodefineasanoncogenicmutatorKentsisandcollaboratorsusedAFA

forefficientandreproduciblecelllysisproteinextractionandchromatinshearing

bull Acoustictechnology-assistedrapidproteolysisforhigh-throughputproteomeanalysisKimetalANALYTICALSCIENCEamp

TECHNOLOGYVol24No6510-5182011DOI105806AST2011246510

- Thispapershowshowcontrolledacousticswavelengthallowsforfasterandmoreefficientdigestionofproteinswithtrypsin

bull EnhancedTrypticDigestioninunder20minutesusingAFAtradeTechnologyIIsaacetalHUPOposter-httpswwwcovariscom

wpwp-contentuploads202007ASMS_2020_Posterpdf

- ThisposterdetailsnumeroustestscomparingtrypsindigestionprotocolshighlightinghowAFAcanincreaseefficiencywhilespeedingthe

processdownto20minutes

bullDevelopmentofanAutomatedHigh-throughputSamplePreparationProtocolforProteomicsAnalysisAruletalBULLETINOF

THEKOREANCHEMICALSOCIETYVolume36Issue7July20151791-1798DOI101002bkcs10338

- TheauthorsoptimizedthecleanupstepsdownstreamofproteinextractionmadeusingcryoprepandAFAacousticultrasonication

bull Label-freequantitativeproteomicanalysisofhumanperiodontalligamentstemcellsbyhigh-resolutionmassspectrometry

HanetalJPeriodontRes20181ndash10DOI101111jre12604

- AFAisusedinthispapertogentlyprocessvariousstemcellpopulations

bull Assessmentofadaptivefocusedacousticsversusmanualvortexfreeze-thawforintracellularmetaboliteextractionfrom

StreptomyceslividansproducingrecombinantproteinsusingGC-MSandmulti-blockprincipalcomponentanalysis

KassamaetalAnalyst2010May135(5)934-42DOI101039b918163f

- ThisstudycomparestheefficiencyofultrasonicAFAandmanualvortexfreeze-thawextractiontechniquesforcomparativemetabolite

profilingofmousetumournecrosisfactoralpha(mTNF-a)expressioninSlividans

bull ShotgunLipidomicsCombinedwithLaserCaptureMicrodissectionaTooltoAnalyzeHistologicalZonesinCryosectionsof

TissuesOKnittelfelderetalAnalChem2018Jul30DOI101021acsanalchem8b02004

- Authorswantedtoanalyzelipidscontents(lipidomes)afterLCMonmouselivertissuesandusedfocusedultrasonicationinthefirst

12

preparationsteps

bull Westernblotanalysisofcellsencapsulatedinself-assemblingpeptidehydrogelsKABurgessetalBioTechniques63253-260

(December2017)DOI102144000114617

- WhenitcomestosolubilizationAFAisthemethodofchoiceasdescribedinthispaperaboutvellsencapsulatedinSAPHs

bull ANon-catalyticFunctionofSETD1ARegulatesCyclinKandtheDNADamageResponseTHoshiietal2018Cell1721007ndash

1021DOI101016jcell201801032

- TheauthorsusedAFAforcelllysispriortowesternblottingandchromatinshearinginChIPexperiments

bull PeptidomimeticblockadeofMYBinacutemyeloidleukemiaRamaswamyetalNATURECOMMUNICATIONS|(2018)9110

DOI101038s41467-017-02618-6

- UseofAFAforsamplepreparationpriortowesternblottingandChIP-relatedexperiments

bull DirectMeasurementofIntracellularCompoundConcentrationbyRapidFireMassSpectrometryOffersInsightsintoCell

PermeabilityLJGordonetalJBiomolScreen2016Feb21(2)156-64DOI1011771087057115604141

- AFAwasusedtolysecellswithinalargerassayintendedforimprovingdrugdevelopment

bull ComparisonofbiochemicalandbiologicaleffectsofML858(salinosporamideA)andbortezomibWilliamsonetalMolCancer

Ther20065(12)3052ndash61DOIMolCancerTher20065(12)3052ndash61

- AuthorsstudycomplexnaturalproductsthathaveantibioticandantiproliferativeactivitieslikesalinosporamideAwhicheffectislinked

toitsabilitytoinhibittheproteasomeBiochemicalandbiologicalactivitiesareassessedcomparedtoaknownmolecule(bortezomib)using

cell-basedreporterstabilizationassaysTumorandbraintissuesareusedasmodels

Information subject to change without notice For research only Not for use in diagnostic procedures

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