BIOLOGY & MEDICINE_Cell Research The Protein Puzzle The human body consists of tens of thousands of proteins. What’s more, these occur in several variants whose concentration in the organism can change over time. Matthias Mann from the Max Planck Institute of Biochemistry 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 proteome – that is, the full set of proteins in the human body – 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 genes Martinsried is as tidy as vert fat into energy, for example, or that encode the blueprints for around a hospital intensive care make oxygen 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 water content from body weight, that these proteins occur in many vari- view,M and several identical-looking most life forms consist of 50 percent ants. Once a gene 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 turn, 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 acid molecule chain that folds things are getting really interesting!” lifetime, the protein composition varies into complex three-dimensional pat- The complex nature of protein analysis according to cell type. Some proteins terns. Some – like keratins in skin and became evident when the human ge- occur in large quantities and in every hair cells – serve as structural substanc- nome was sequenced in 2001. The sci- cell, while others occur only in trace es. Other proteins, known as enzymes, entists working on the Human Genome amounts and only in certain tissues. > Photo: Axel Griesch 54 MaxPlanckResearch 3 | 17 Nothing happens in proteomics without automation. Robots greatly facilitate the task of pipetting, allowing researchers to analyze tens of thousands of proteomes. Photo: Axel Griesch xxxxxxxx 3 | 17 MaxPlanckResearch 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 trypsin, a tissue as well as how it changes,” known constituents, for instance to de- a digestive enzyme, they snipped pro- Mann says. tect toxins 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 peptides 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 emergence 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 peptide 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- structures of proteins collapse when trometry with ionization is too harsh a national databases. In addition to the Photo: Axel Griesch 56 MaxPlanckResearch 3 | 17 BIOLOGY & MEDICINE_Cell Research A section of the protein spectrum of a cancer cell: The proteins are first cut into peptides of varying molecular weight (shown in different colors), then separated from each other in chromatographic columns and subsequently analyzed in a mass spectrometer. molecular weights of every conceivable Mann miniaturized the tubes used in proteome of an entire organism, iden- peptide, the databases contain informa- this type of chromatography down to tifying the 4,399 proteins in a yeast tion about the protein to which each a diameter of just a few nanometers. fungus. He and his Max Planck col- fragment belongs. MaxQuant compares With these tubes, he needs only min- league Frank Schnorrer achieved their the data from the mass spectrometer ute quantities of a sample: “Our nano- latest breakthrough in 2016, when they with the content of the databases and chromatography enables us to obtain decoded the fruit fly proteome. They reconstructs the protein composition of sufficiently high concentrations of pro- discovered that the tiny insect has a sample from the results. teins that are present in the sample in around 10,000 proteins. “By compari- vanishingly small amounts.” son, there are around 13,000 proteins CHROMATOGRAPHY IN By combining electrospray ioniza- in the mouse brain,” Mann explains, MINIATURE FORMAT tion, MaxQuant and nanochromatog- and only 10 percent of them are limit- raphy, Mann succeeded in doing what ed to specific cell types. Nevertheless, electrospray ionization had previously seemed impossible: de- Mann’s method has since become and MaxQuant together were unable to coding an organism’s entire proteome. the standard in proteomics laboratories overcome all the obstacles of protein In 2008, the researcher analyzed the around the world. Without the help of analysis. For example, they could not detect proteins that are present in a sample only in trace amounts. This was because the researchers needed a rela- tively large liquid sample to separate the proteins from the other compo- nents by chromatography before the proteins are analyzed in a mass spec- trometer. As a result, the trace proteins were excessively diluted and could no longer be detected. The electrically neutral proteins are first cut into smaller peptide fragments. In order for the peptides to be deflected along different trajectories by the electrical field of the mass spectrometer, they must be electrically charged. This is accomplished by the electro­ spray ionization method, in which the pep­ tides are given an electric charge in a metal capillary tube and then sprayed out at the tip of the tube. Photo: Axel Griesch Photos: MPI of Biochemistry (top), Axel Griesch (bottom) 3 | 17 MaxPlanckResearch 57 BIOLOGY & MEDICINE_Cell Research as many samples as possible. A year Mann was able to show that an organ- from now, we expect to be able to ana- ism’s day-night rhythm crucially de- lyze 100 proteomes a day,” Matthias pends on the phosphorylation state. Mann predicts. Researchers will then be “There are an enormous number of able to study, for example, how a pa- protein variants whose importance we tient’s protein concentration changes still don’t understand.