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The Protein Puzzle

The Protein Puzzle

& MEDICINE_Cell Research

The Puzzle

The body consists of tens of thousands of . What’s more, these occur in several variants whose concentration in the can change over time. Matthias Mann from the Max Planck Institute of in Martinsried therefore needs clever algorithms and a lot of computing power for his research. His goal, after all, is to decode the entire human – that is, the full set of proteins in the – for the benefit of medical science.

TEXT TIM SCHRÖDER

atthias Mann’s lab in speed up metabolic reactions: they con- Project identified around 20,000 Martinsried is as tidy as vert into , for example, or that the blueprints for around a hospital intensive care make available to cells as a the same number of proteins. unit. The glass walls and source of energy. If we subtract the Only gradually did it become clear doors provide a clear body content from body weight, that these proteins occur in many vari- view, M and several identical-looking most forms consist of 50 percent ants. Once a has been read, parts workstations are set up around the protein. Without proteins, there would of the messenger RNA, which serves as room. At each station, a robotic arm be no life on earth. a protein template, can be cut out. hanging from the ceiling picks up small This gives rise to RNA molecules of plates containing samples and places A MATURE TECHNOLOGY various lengths, each of which is trans- them in the instruments. Technically lated into a different protein. Numer- speaking, these systems represent the Matthias Mann initially studied phys- ous proteins, in , must be trimmed essence of what Matthias Mann has ics and mathematics, but he has been before they can be used as finished been developing over the years: ma- investigating the world of proteins molecules, while chemical tags are ap- chines capable of processing and ana- since the 1980s. “It has taken two de- pended to others. All in all, there are lyzing proteins at breathtaking speed. cades to develop the technology that hundreds of thousands of protein vari- Mann is interested in these biomol- allows us to analyze proteins in a rea- ants that interact in a finely orches- ecules because they are involved in al- sonable manner,” he says. “We’ve trated choreography. most all of an organism’s biological reached the point where we can actual- In addition, whereas an organism processes. Proteins are made up of an ly apply the technology − and now possesses the same genes throughout its amino molecule chain that folds things are getting really interesting!” lifetime, the protein composition varies into complex three-dimensional pat- The complex of protein analysis according to type. Some proteins . Some – like in and became evident when the human ge- occur in large quantities and in every cells – serve as structural substanc- nome was sequenced in 2001. The sci- cell, while others occur only in trace

es. Other proteins, known as , entists working on the Human amounts and only in certain tissues. > Photo: Axel Griesch

54 MaxPlanckResearch 3 | 17 Photo: Axel Griesch

xxxxxxxx researchers to analyze tens of thousands of . proteomes. of thousands of tens analyze to researchers allowing pipetting, of task the facilitate greatly Robots automation. without in happens Nothing 3 | MaxPlanckResearch 17MaxPlanckResearch 55 Under the watchful eye of Heiner Koch, researchers Florian Meier and Scarlet Beck (left to right) analyze protein samples using the six mass spectrometers set up in Matthias Mann’s (far right) laboratory alone.

Different proteins are active depending they are heated or mechanically stressed, method for the sensitive proteins. In on the tasks the metabolic machinery and they clump together. It was there- the lab of his doctoral supervisor, John is carrying out. “If we want to know fore not possible to analyze proteins B. Fenn, at Yale University, Matthias how the metabolic machinery works or with the conventional method of mass Mann, together with other colleagues, what its momentary state is, we must spectrometry. Mass spectrometers are developed a gentler solution in the be able to analyze the protein profile in used to analyze samples containing un- early 1980s. With the help of , a as well as how it changes,” known constituents, for instance to de- a digestive , they snipped pro- Mann says. tect in tissue samples. teins into approximately ten-amino- Scientists have always realized how acid-long fragments – so-called pep- important proteins are for body pro- DEFLECTION IN AN ELECTRIC FIELD tides. They then sprayed the cesses. Now, however, a growing num- through a fine tube, imparting an elec- ber of researchers have become inter- Before proteins can be investigated in a trical charge to them. Using this electro- ested in studying the complete set of mass spectrometer, they must be con- spray ionization method, they were proteins in the body. This eventually re- verted into charged particles, for exam- able to analyze proteins for the first sulted in the of the research ple by bombarding them with electrons time in a mass spectrometer – a method area we now call proteomics. But enor- or other charged particles, which con- for which Fenn was awarded the Nobel mous quantities of data are required to verts them into electrically charged Prize in Chemistry in 2002. analyze the protein composition of any ions. Only electrically charged mole- The time had now come for com- life form. A huge volume of data must cules are deflected from their path puter scientists, as it is almost impossi- also be collected for evaluating and in- through the electric field of the mass ble for a human being to deduce the terpreting the results. Proteomics thus spectrometer. The amount they are de- original proteins from a mix. relies on sophisticated data processing. flected depends on their charge magni- Together with his colleague Jürgen Cox, A major problem in analyzing pro- tude and molecular weight, and this in- Mann developed an analytical program teins is that they are very sensitive. As formation allows scientists to infer a called MaxQuant, which can compare anyone who has ever boiled or beaten molecule’s identity. many thousands of peptide fragments an egg knows, the three-dimensional However, conventional mass spec- with information contained in inter-

of proteins collapse when trometry with ionization is too harsh a national databases. In addition to the Photo: Axel Griesch

56 MaxPlanckResearch 3 | 17 Photo: Axel Griesch

Photos: MPI of Biochemistry (top), Axel Griesch (bottom) of the tube. the of tip the at out sprayed then and tube capillary metal a in charge electric an given are tides pep the which in method, ionization spray electro the by accomplished is This charged. electrically be must they spectrometer, mass the of field electrical the by trajectories different along deflected be to peptides the for order In fragments. peptide smaller into cut first are proteins neutral electrically The longer bedetected. were excessivelydilutedandcouldno trometer. Asaresult,thetraceproteins proteins areanalyzedinamassspec nents bychromatographybeforethe the proteinsfromothercompo- tively largeliquidsampletoseparate because theresearchers neededarela- sample onlyintraceamounts.Thiswas detect proteinsthatarepresentina analysis. Forexample,theycouldnot overcome all theobstaclesofprotein and MaxQuanttogetherwereunableto Nevertheless, electrosprayionization MINIATURE FORMAT IN a samplefromtheresults. reconstructs the protein composition of with thecontentofdatabasesand the datafrommassspectrometer fragment belongs.MaxQuantcompares tion abouttheproteintowhicheach peptide, the databasescontaininforma conceivable weightsofevery molecular ­ ­ - - this typeofchromatographydownto Mann miniaturizedthetubesusedin In 2008,theresearcher analyzedthe coding anorganism’s entireproteome. had previouslyseemedimpossible:de- raphy, Mannsucceededindoingwhat tion, MaxQuantandnanochromatog- vanishingly smallamounts.” teins thatarepresentinthesample sufficiently highconcentrationsofpro- chromatography enablesustoobtain ute quantitiesofasample:“Ournano- With thesetubes,heneeds only min- a diameterofjustfewnanometers. By combining electrospray ioniza- around the world. Without the help of the standardinproteomicslaboratories ed tospecificcelltypes. and only10percent ofthemarelimit- in themousebrain,”Mannexplains, , therearearound13,000proteins around 10,000 proteins. “By compari- discovered thatthetinyinsecthas decoded thefruitflyproteome.They latest breakthroughin2016,whenthey league FrankSchnorrerachievedtheir . HeandhisMaxPlanckcol tifying the4,399proteinsinayeast proteome ofanentireorganism,iden- analyzed in a mass spectrometer. mass a in analyzed subsequently and columns chromatographic in other each from separated then colors), different in (shown weight molecular varying of peptides into cut first are proteins The cell: a of spectrum protein the of section A BIOLOGY &MEDICINE _Cell Research Mann’s methodhassincebecome 3 | MaxPlanckResearch 17MaxPlanckResearch 57 - 58 tem’s workflowtoallowusanalyze “We’re currently optimizingoursys ods havetobespeededupeven more. find applicationinhospitals, themeth tein changeswithtime. how theconcentrationofaspecificpro a patient canbe compared to determine a sample.Thus,multiplesamplesfrom ty, butalsothequantityofproteinsin can nowdeterminenotjusttheidenti- the MaxQuantsoftwaresothatthey thinkable. CoxandMannhaverefined computers, itsusewouldstillbeun functions. specific their out carry to repertoire protein different completely a use often they genes, of ments comple identical have cells all Although (red). mode reproductive the by unaffected are others Still (). asexual during mainly produced are others while (blue), reproduction duringamplified sexual are attractants, protein as such proteins, Some modes. reproductive both in proteins of ratios the shows graph The asexually. and sexually both reproduce can organism cellular uni­ The analyzed. was proteome complete whose organism first the was Baker’s BIOLOGY &MEDICINE _Cell Research MaxPlanckResearch 3 MaxPlanckResearch However, beforeproteomics can | 17 ­

- - - - as . phosphate molecule–aprocess known are activatedbytaggingthem witha dismantled. Moreover, manyproteins during whichtheproteinis gradually teins, initiatingabreakdown process ple, bindstoagingordefectivepro cur in.Theproteinubiquitin,forexam- the manyvariantsthatproteinscanoc the same. responds other words,noteveryone even iftheyfollowthesamediet.In crease atthesamerateineveryone, proteins don’tthat inflammatory de- the MaxQuantsoftware.Theyfound proteinsusing quantity ofinflammatory 1,000 proteomesanddeterminedthe his colleaguesanalyzedmorethan all patients during a . Mann and proteins decreasesthesameamountin whether thequantityofinflammatory tions. The researchers wanted to know reac- that aretypicalofinflammatory an ,producingproteins The bodyreactstoobesityasifitwere TO ADIETIN THE SAMEWAY NOT EVERYONE RESPONDS an eight-weekdiet. proteins ofobesepeoplechangeduring University Hospital,studiedhowthe together withdoctorsatCopenhagen and healthypatients.To thisend,Mann, differences inthemetabolismofsick compare groupsofpeopletodetermine ease progresses. during thecourseofadayorasdis- tient’s proteinconcentrationchanges able tostudy, forexample,howapa Mann predicts.Researchers willthenbe lyze 100proteomesaday,” Matthias from now, weexpecttobeableana- as manysamplespossible.Ayear Proteome analysisiscomplicatedby The scientistsarealreadyableto - - - Mann wasabletoshowthatanorgan- changes inapatient’s proteomethat ferent states. But itis precisely these tire groupsofproteinscantakeondif- still don’t understand. Moreover, en protein variants whose importance we “There areanenormousnumberof state. pends onthephosphorylation ism’s day-nightrhythmcruciallyde protein is released. Such minute amounts protein isreleased.Suchminute amounts littleofthetelltale meaning thatvery smallinthe early stage, is still very cer in the proteome because the tumor find early signs of malignant skin can- cy. It would be extremely difficult to treatment ofdiseasesisstillinitsinfan diseases intoaccount. also takes existing knowledge about production ofcertainproteins.Perseus when themetabolic system increases proteins occurandwhatitmeans proteins −forexample,wherespecific mulat tional databasescontainingtheaccu- ysis. Thesoftwareaccessesinterna- MaxQuant toconductabigdataanal uses thestatisticalproteindatafrom help: Perseus,astheprogramisdubbed, colleague JürgenCoxisexpectedto Mann says. ofhisorherhealthstatus,” overview on anindividual’s proteometogainan future, wewillrelymuchmoreheavily know, though,thatisn’t enough.Inthe single protein. Based on what we now tests showthepresenceoramountofa versial owingtoitsunreliability. “Such of cancer, but which is contro- PSA level, which can be an indication day’s diagnostictests. littleofsometo fore thinksvery diseases,” saysthescientist,whothere are decisivewhenitcomestotreating As anexample,hementionsthe The proteome-baseddiagnosisand Another programdevelopedbyhis ed fundofknowledgeabout ------

Graphic: Max Planck Institute of Biochemistry/Matthias Mann Graphic: Max Planck Institute of Biochemistry/Matthias Mann

Photo: Axel Griesch

medical science. teomics forthebenefitofbiology and now help harness the potential of pro- his research raised. As a result, he can terest inthebiologicalquestions that physics, andinsteadtookan earlyin- clusively on computer science and fortunate itwasthathedidn’t focusex the ground,”saysMatthiasMann.How things are only now really getting off but brought usagiantstepforward, word. “Ournewtechniqueshave term proteomicsbecameamerebuzz- disappeared fromthescene,and plaints soonfollowed.Manystartups less forroutineclinicalpractice.Com- the timewasutterlyinadequate. ization, it’s clear that the technology of chromatography andelectrosprayion- ers. In lightof such methods as nano- toclinicalresearchanalysis asaservice up companiesbeganofferingproteome nome wasdecodedin2001,manystart of themillennium.Afterhumange ly advancedremarkablysincetheturn help ofnanochromatography. can’t be reliably detected even with the Pavel Sinitcyn and Art Carlson – and playing a relaxing game of chess during a break. break. a during chess of game relaxing a playing and – Carlson Art and Duitama, Camila Sinitcyn Cox, Pavel Jürgen Rudolph, Jan left): front from (clockwise computer the on data Analyzing Accordingly, theresultsproveduse- Nevertheless, proteomicshasclear-

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l l l TOPOINT THE GLOSSARY time. Cells have different proteomes depending on their cell type. There are thus at at thus are least There type. cell their on depending proteomes different have Cells time. 400,000 Proteome: gene. single a to traced be can different proteins ten to up , In gene. single a from produced are proteins pro multiple several teins, into cleaved subsequently is chain acid amino an when or encodes proteins, gene a several When proteins. of the alter to added subsequently and are phosphate residues, as such tags, molecular Small products. gene a in different of resulting number moved, or out cut are molecule RNA messenger a of sections for splicing, instance, alternative as known process a In (). been into has RNA converted messenger after or () read been has gene a from after results changes diversity enormous This genes. its of number the than greater times several be can produces organism an proteins of number Protein modifications: The also affected by environmental influences such as medications and pollutants. and medications as such influences environmental by affected is also composition protein The health. of state and diet age, organism’s produced an be on may depending proteins different example, For factors. many on depends proteome The    proteins indiseaseprocesses. the occurrence andfunctionofproteins. This shedslightontherole ofindividual Using thePerseusprogr about peptidesandpr The MaxQuant program asrepositories accessesdatabasesthatserve ofknowledge Only sophisticatedcomputeralgorithmscanr For proteome analysis, theproteins mustfirstbe cut upintopeptidefragments. from thehugevolumes ofdatagenerated. 250 proteins. However, they aren’t all produced by all the body’s cells at any given given any at cells body’s the all by produced all aren’t they However, proteins. different proteomes corresponding to the the to corresponding proteomes different 80,000 between contains body human the that estimated now is It oteins. am, researchers analyze information from databaseson econstruct theoriginalproteins 250 cell types in the human body. 3 | MaxPlanckResearch 17MaxPlanckResearch and and

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