eduardo cesarSensitivity and high resolution: the LNLS’s mass spectrometers allow for the identification of the amino acid sequenceeduardo cesar
This year the Nobel Prize for Chemistry was awarded to two medical doctors and a biochemist who discovered how cells break down and reuse their old or defective proteins. During 2002, a chemist and an engineer shared another Nobel Chemistry Prize for having improved two techniques that had allowed for the analysis of proteins, using the mass spectrometer, today essential in this area. It is not by chance that one of the highest honors in science in the world had recently recognized, twice, the value of the study of these molecules, abundant in any microorganism, plant or animal.
Over the last five years, after the sequencing of the genomes of almost one hundred and fifty organisms, the identification of the structure, function and the means of interaction of these molecules, coded by genes, has become a world priority, as it represents an apparent pathway in understanding, with greater detail, the chemical reactions that keep organisms alive or that make them perish. From this knowledge, it is hoped to obtain more efficient ways of combating illnesses – a simple cold or an agricultural pest – or even of prolonging life.
This is an immense world whose exploration has hardly begun. The Protein Data Bank, a data base specific to proteins, stores information dealing with the structure of approximately 25,000 of these molecules of plants, animals and microorganisms. This is small when compared with, for example, the number of human proteins, estimated at between 100,000 and 1 million. Today a week does not go without proteins being the highlight in top class scientific magazines ? for example, in the middle of September twenty of the fifty one studies published in the Proceedings of the National Academy of Sciences covered these molecules directly or indirectly.
Even without a unifying project such as the Human Genome Project that brought together dozens of laboratories in the sequencing of the genetic material of our species, the study of proteins is advancing rapidly in Europe and the United States – and also in Brazil. Here there already are around two hundred research groups in this area, denominated proteomics, which have gained an impulse with the entrance into operation of two new pieces of equipment at the National Light Synchrotron Laboratory (LNLS), in Campinas.
With these new machines, which determine the sequence of the blocks make up by the protein, the amino acids, Brazil has gone on to become a member of the select team of countries with the technology to analyze in detail the structure of proteins. Installed in July of 2003, the LNLS’s new pieces of equipment – two mass spectrometers acquired for US$ 1.3 million, financed by FAPESP – were liberated in September for research groups from any state in the country, assuming that the work proposals areapproved by the LNLS and the results shared by other teams.
Those selected
From the first batch of fifty one proposals for the use of the equipment, the LNLS selected twenty, made up of research groups from four states – Sao Paulo, Rio de Janeiro, Ceará and Rio Grande do Sul. These are projects dedicated to the analysis of proteins of the microorganisms that cause diseases in plants, such as Xylella fastidiosa, which attacks orange groves, or in animals, as is the case with the bacterium Mycoplasma hyopneumoniae, one of the causes of pneumonia; of Leptospira interrogans, the agent for leptospirosis; and the protozoa Trypanosoma cruzi, responsible for Chagas’s disease. The selected teams have until December to also investigate the proteins associated with the development of tumors and the activation and deactivation of genes, (see the list at: www.revistapesquisa2.fapesp.br). In January the LNLS will launch a notice for the selection of the second batch of proposals.
“Evidently we don’t find ourselves at the same level of countries such as the United States or the United Kingdom in which the use of mass spectrometers is highly developed and well spread, but we are the pioneers in Latin America in protein research”, comments the biochemist Rogerio Meneghini, who directed the LNLS’s Structural Biology Center until February of this year and today is the coordinator of the laboratory projects.
“Our objective is to consolidate or to form groups of excellence in proteomics, in the same manner as there are today first class teams in genomics in Brazil.” According to him, of all of the groups in this area in the country, close to forty within a few years, will be in condition to compete internationally with discoveries relevant to the structure of proteins, the watershed that explains how these molecules interact among themselves and with others. This is a number similar to the laboratories today qualified to carry out the sequencing and analysis of genes.
It is easy to understand why researchers find themselves attracted by proteins, whose importance goes well beyond common sense – that they are the main components of foods such as meat, soy and milk. It is these molecules that form and keep the cells and the tissues of human beings working. Proteins are found in cells and tissues in considerable quantities when compared to other types of molecules: for example, they correspond to around 30% of muscular mass or of the liver. Their roles vary according to the situation and the location in which they find themselves. Proteins can act as transporters and, like luggage porters at airports, can take compounds from the outside to the inside of cells, enveloped by membranes made up of lipids, sugars, and other types of proteins.
At other times they function as a type on antenna, capturing information sent by neighboring cells. They also participate in chemical reactions that result in the production of energy, in the formation of memory, indeed, in the control of the organism as a whole. They are the laborers – always alert – of human beings. In a situation of danger, it is a protein that functions as a hormone, adrenalin, which makes the heart beat faster, feeding the muscles with more blood and thus leaving the body prepared to fight or to flee.
It wasn’t right now that Brazilian researchers jumped into this labyrinth. Over the past five years, national laboratories began to import the first mass spectrometers, which now total dozens in the country. They can be found at laboratories such as that of the biologist Carlos Bloch Junior, from the Brazilian Agricultural Research Corporation (Embrapa) in Brasilia, of the chemist Mario Sergio Palma, from the São Paulo State University of Sao Paulo (USP) in Ribeirão Preto.
Also with this type of equipment are the biochemists Antonio Carlos de Camargo, from the Butantan Institute and José Camillo Novello, from the State University of Campinas. At USP in São Paulo, the pharmacologist Gilberto De Nucci and the parasitologist Igor de Almeida have mass spectrometers They also exist at the laboratories of the biophysicists Luiz Juliano Neto, from the Federal University of Sao Paulo, and Paulo Bisch, from the Federal University of Rio de Janeiro.
“These first pieces of equipment are powerful, but the sensitivity and the accuracy of mass spectrometers for the study of proteins is increasing on a daily basis”, explains Meneghini. In his opinion, the new pieces of equipment at the LNLS will allow for the study of larger proteins, with a greater possibility of determining the sequence of amino acids of which they are constituted.
Complementary action
However, in order to make this jump Meneghini and Bloch worked for close to a year on making a choice, purchasing and assembling the spectroscopes. They adopted three basic criteria: the equipment must have high sensitivity in order to detect proteins contained in samples with billionths of a gram of biological material; present a resolution that would make the identification of each one of the amino acids possible, though they have masses close to each other; and provide the results rapidly – one of the machines analyzes a thousand samples per hour.
“Still back at the phase of selection”, Bloch tells, “we took protein samples of the bacterium Xanthomonas citri to be tested by four mass spectrometer manufacturers who have representatives in the country, in order to make comparisons on the sensitivity and precision of their equipment”. And it was Bloch himself who carried out the first scientific study using the new machines: an analysis of the protein hylaseptinea P1. Extracted from the secretion of the hair of Hyla punctata, a green and lively tree frog found in the Amazon, the hylaseptinea acts against the bacteria that cause hospital infections such as Staphylococcus aureus and Pseudomonas aeruginosa, or a fungus, the Candida albicans, which shows up in immune depressed people, as demonstrated in a study published in March of this year in the Journal of Biological Chemistry.
The LNLS’s two pieces of equipment are slightly different – the advantage is that one complements the reading of the other. One of them applies an electrical discharge to the proteins and fragments them into electrically charged parts, which are then identified in accordance with their mass. This is a technique known as Electrospray Q/TOF, used in the study of molecules that are water soluble such as hemoglobin, the protein that transports oxygen and gives the red color to blood.
The other piece of equipment discharges a laser beam upon the proteins stored in a crystal, and thus they become electrically charges. By way of this technique, called Maldi-TOF/TOF, the structures of proteins found in cell membranes can be evaluated. “To understand the structure of these molecules is essential to finding out new ways of combating various diseases, since the membrane of a parasite functions as its sensorial organ and allows, for example, that it recognizes its host cell”, explains Bloch.
The main advantage in relation to the existing mass spectrometers in the country is that the recently installed machines in Campinas – under the care and protection of the chemist Fabio Cesar Gozzo, the LNLS’s Mass Spectrometer Laboratory coordinator – identify each one of the amino acids that make up the protein and the sequence in which they fit together in order to form it. In this manner, it could well become easy, for example, to design molecules of medicines that fit with precision into a determined protein and prevent the occurrence of a cancer or the action of a bacterium such as Xylella fastidiosa or the Xanthomonas citri, today viewed as orange tree pests. This would be an advance and then some more. “It is as if up until now we attempted to mount a jigsaw puzzle with our eyes closed, feeling about in the dark to fit together a piece here and another there, and trying to verify if a medication works in combating a determined disease”, compared Glaucius Oliva, the coordinator at the Physics Institute of USP in Sao Carlos and the Director of the Structural Molecular Biology Center, one of the ten Research, Innovation and Diffusion Centers financed by FAPESP. With the structure of proteins in their hands, the researchers will go on to work without a mask over their eyes.
But many are reluctant to dive into the world of proteins. It’s not everyone’s cup of tea. “As interesting as it is, biologists consider the theme to be more than just complex, whilst chemists believe that the proteins are excessively large”, commented Dr. Bloch. The challenge intimidates even the most experienced, perhaps for being even greater than that confronted up until this moment with the sequencing of various genomes.Although the genes contain the recipes of the proteins, to understand the grouping of genes – the genome – of an organism is not sufficient to know how they are nor how they act. As well as this, each gene can originate more than one protein.
Distinct structures
They are things that are very different. Genes are specific strips of molecular – deoxyribonucleic acid, the genetic material of cells. They take the form of long sequences of four small molecules known by the letters A, T, C and G (adenine, thymine, cytosine and guanine respectively). Now the proteins are much more complex, composed of long sequences of twenty different types of amino acids, resulting in a grouping of dozens to thousands of units – insulin, an enzyme that facilitates the entrance of sugar into the cells, is made up of only 51 amino acids, whilst myosin, one of the main muscle proteins, groups together into its structure close to 1,800 of these blocks.
Another fundamental distinction: whilst the DNA molecule always takes the form of a spiral stairway or a double helix, as was discovered by James Watson and Francis Crick in 1953, proteins can have forms that are very distinct – varying from a small globe to a boomerang, for example. There is a further complication: no sooner do they leave the interior of the cells, where they are manufactured, than the proteins associate themselves to sugars and fats, forming even greater complexes – the glycoprotein CD 44 functions as a type of cellular cement, maintaining the cells close to each other. In the case of proteins, this three dimensional structure makes up the difference, since its form is directly linked to the function that it is capable of exercising.
The Project
Proteomics Studies in Sao Paulo State; Modality Regular Line of Research Assistance; Coordinator Fabio Cesar Gozzo – LNLS; Investment R$ 5,391,153.26
