M1D3:Analyzesmallmoleculemicroarray(SMM)data1. BECommunicationLabworkshop2. Prelab discussion3. CompleteSMMdataanalysis4. SelectligandsfromidentifiedSMMhits

02/15/19

• Homeworkdueatstartoflaboratoryinboxonfrontbench

• Carefullyreadallprotocolsteps• FirstquizscheduleforM1D4

OverviewofMod1experiments

Butfirst,decipheringyourdigestresults

Otherpotentialexplanations

• Smear?• Incompleteand/ornocutting?• Extrabands?

QuickrecapofSp17

Usingimmunofluorescencetodetectligandbinding

Howarecompoundsimmobilized(printed)onanSMMslide?

Leiferetal.MethodsinEnzymology.2018.

WhatcompoundsareprintedonanSMMslide?

Leiferetal.MethodsinEnzymology.2018.

SMMquantificationsteps1. AlignGALfiletofluorescenceon532nmchannel

(sentinelspots)2. Quantifyfluorescenceon635nmchannel3. Identify‘hits’withimprobablyhighfluorescence4. Identifycompoundsthathitrepeatedly5. Comparetophitstocommonbinderslist

Imagesrepresentarraysofnumbers• Eachpixelisa16-bitnumberthatrepresentsintensity• Computationalanalysisusedtodefine‘hits’

AlignSMMresultstousingsentinelspotsEveryspotcanbelocatedusingintersectinglines

betweensentinels

Quantifyfluorescencetoidentifyhits• Foreground

• Background

Signal-to-noiseratio(SNR)=μforeground - μbackgroundσbackground

HowtoevaluatetheSMMresults• Isthebackgroundnoisy?• Arethepositivecontrolseasilyrecognized?• Doanyareasappearstrange?Damaged?• Manufacturerorhandlingdefects

• Arethehitsalignedwithprintingspots?• Doyoutrustthedata?

CalculaterobustZscoretoidentifyhits

RobustZscore= SNRi-median(SNR)

median(|SNRi-median(SNR)|)*1.48

medianabsolutedeviation(MAD)

scalefactorforthenormaldistribution

RobustZscoreseliminatetheinfluenceofoutliers

Whatdoweexpecttosee?

Factorsthatinfluencehitidentification

• Howmanyfalsepositivesareexpected?• Morehitsneededifconfidenceislow

• Howmanychemical‘patterns’areevident?• Repeatedpatternsbetweencompoundsmayincreaseconfidence

• Arethehitsuniquetothescreen?• Promiscuousbindersmaydecreaseconfidence

Fortoday…• Besuretoselectligandsfromthesign-upsheetatthefrontlaboratorybench• Eachteamwilltesttwoligands

ForM1D4…• Draftoverviewschematic(image,title,caption)• Outlineintroductionusingtopicsentences– Includereferenceinformation!!!

Notesonoverviewschematics…AdaywithNoreen:

Wakeupat6aandbrushteeth

Eatbreakfastanddrinkcoffeewhilewatching

thelocalnews

Getreadyforthedaybyfixinghairandmakeup

WalkandfeeddoggosCheck,respondtoemailsandreviewlectureslides

TeachW/Flaboratory

sectionfrom1-5pin56-322

Gotogymandrun1mile,

practicestrengthtraining,

completeWODMakeandeatdinnerBrushteethandgoto

bedat10pTakethetrainhome

WalktoworkatMIT

Notesonoverviewschematics…AdaywithNoreen:

Figure1:SchematicimageofwhatNoreendidtoday.Heralarmclockwentoffat6aandshesnoozedfor9minutes,thenshebrushedherteeth.Forbreakfastsheateoatmealwithblueberriesandpepitas.Shewalkedherdogs,CalyandBuddy,aroundtheblockthenfedthemchickenandrice….Atthebox,sheran1mileontheAirTreadthen…

Notesonoverviewschematics…AdaywithNoreen:

Wakeupandgetreadyfortheday

CompleteWOD

Gohomeandgetreadyforbed

Gotowork

Yay20.109!!

Figure1:AverageweekdayforNoreen.OverthecourseofanormaldayNoreenisactivefrom6auntil10p.SheworksasanInstructoratMIT.Afterwork,sheexercisesataCommonwealthCrossFit.WOD=workoutoftheday.

Notesontopicsentences:• Firstsentenceofeachparagraph• Should‘funnel’frombigpicturetopictoyourspecificresearchproject• Provideonlythebackgroundneededtounderstandresearch/problem/goal• Clearlystatewhatisnotcurrentlyknown• Addresshowyouwillfillknowledgegap• Providepreviewofyourresults

• Includereferences!!

Specificbackground

KnowledgegapStatementofproblem

Hereweshow…

ImpactStatement

Hypothesis

Howwillyouintroduceyourstory?