Proteomics: Weighing the evidence

Sequencing of the human genome was completed in 2001, a few years ahead of schedule. One of the implicit aims of this project was to identify disease markers. However, to date, we still grapple with the elusive causes of many diseases and the hunt for biomarkers is still on. This is rooted in the fundamental problem that the genome is a mere blueprint for a vastly complex network of interactions orchestrated principally by proteins. The protein complement of the genome is what we refer to as the proteome and the variety of disciplines aiming to survey and understand the proteome fall under the umbrella of proteomics.

The complexity of studying the proteome arises from the fact that proteins encoded by genes can undergo a number of processing events that are regulated in space and time. Although a particular organism is endowed with a single genome, there are several corresponding proteomes for each cell population at a certain time and under specific environmental influences. At the molecular level, a single gene encoding a protein, yields a product that is then processed (e.g. glycosylation, phosphorylation) amplifying the variants. In biological fluids like plasma, it is not uncommon to find extraneous proteins due to tissue leakage in cases of disease. Considering mature forms, degradation products and modified species, numbers could easily escalate to over a million protein products!

The techniques involved in proteomics include those that can be used for protein separation, identification and quantification. More advanced applications allow for structural analysis, mapping modifications, defining complex interactions and even tracking protein regulation in the cellular context. The most common strategies include 1D and 2D gel electrophoresis, various types of chromatography and mass spectrometry (MS). The latter is powerful as a stand-alone technique in proteomics research, but maximal effectiveness is achieved when it is coupled to the above-mentioned separation techniques.

Mass spectrometry, as its name implies, basically measures the "weight" of molecules. It is amazing how much information one can get by merely weighing things. However, when one realizes that MS offers resolution enough to distinguish a mass difference of a single proton (1 Da), and in some cases even as much as resolving a mass difference in the milli-dalton range, then it is not surprising why this technique is so powerful.

Most of the variations we try to track as indicators of abnormal physiology can be related to a change in the mass profile of a sample. One can, for example, easily detect the presence of a "foreign" protein or disappearance of a resident enzyme. When two molecules interact or are transformed, there is a corresponding change in mass (except rearrangement reactions). When peptides or proteins are modified chemically, there would also be a change in mass seen as an increase attributed to the moiety added. Because MS allows directed fragmentation, even isomers can be distinguished based on the different fragments generated as a function of structure; hence, products of different masses are obtained and give insight into chemical structure.

Analytes or samples to be studied by MS can be solid, liquid or gas as starting materials as long as charged analytes can be generated from them, because MS depends on gas-phase mass separation and detection of ions (charged molecules). Soft-ionization methods, namely electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), allowed routine analysis of biomolecules. These developments in MS were so important so that it was awarded the Nobel Prize in Chemistry in 2002.

MALDI coupled to the time-of-flight analyzer has been a workhorse for the strategy called peptide mass fingerprinting. This is based on the fact that proteins having unique sequences also often have distinct patterns of cleavage sites for a given enzyme. The cleaved protein therefore yields peptides that reflect this unique distribution (distinct sequence), allowing the matching of the experimental digest with a theoretical digest stored in databases, thus aiding the identification of a protein. However, this is not a very high-resolution method and it often fails when you are looking at related proteins or isoforms. Therefore, it is still important to get actual sequence information and this can be obtained through de novo sequencing.

De novo sequencing can be achieved using tandem MS. This refers to the possibility of using a mass filter allowing the isolation of ions of a desired mass and subsequently subjecting these to collisions with inert gas molecules to cause fragmentation. In this way, structural information via MS could be obtained from peptides (or small molecules). The weakest bonds happen to be along the peptide bond, making the reading of the sequence from the mass differences of the various fragments rather straightforward. Fragment sizes differ by a discernable mass corresponding to known amino acids from which the sequence can be pieced together. An additional feature of this strategy is that it allows detection of modifications on proteins, for example, when one detects mass differences not corresponding to any of the essential amino acids. Typical modifications may include a mass increase of 80Da for phosphorylation or addition of 16Da for oxidation.

Mass spectrometry indeed is a powerful technique not limited to mere mass measurements for small molecules but could be powerfully employed for the identification, characterization and structural analysis of any biomolecule, particularly proteins. By weighing molecules and their fragments, we could weigh the evidence for potential biomarkers related to disease and for understanding molecular and cellular networks.

* * *

Leopold L. Ilag has a B.S. Biology degree (magna cum laude), major in Genetics from UP Los Baños. He obtained his Ph.D. in medical biochemistry and biophysics (molecular neurobiology) from the Karolinska Institute in Stockholm, Sweden. As a postdoctoral fellow of the European Molecular Biology Organization (EMBO), he worked on nuclear magnetic resonance spectroscopy of biomembranes and peptides at the Max Planck Institute for Biochemistry in Martinsried, Germany. He trained in biomolecular mass spectrometry at the University of Oxford and the University of Cambridge, UK. Currently he is an associate professor at the Department of Analytical Chemistry, Stockholm University and is the director of the Stockholm University Proteomics Facility. Log on to http://www.anchem.su.se/staff.asp. E-mail him at [email protected].