Current Analytical Chemistry - Volume 5, Issue 2, 2009

Over the last twenty years, biomolecular scientists learned to switch from the model to the result, from a design based on a four letter alphabet to the final twenty letters based outcome of the genetic project. From genome to the proteome. This important step was taken because the evolutive information is carried by a very rigid molecule, the DNA, and becomes effective in much more flexible objects, the proteins, whose chemical and physical freedom supports every life process. “Proteomics” is a relatively newborn science, whose aim is to study the entire complement of proteins produced by an organism. No other science has attracted such an impressive amount of human, technological and economic resources among the large field of biochemical research over the last decade, because proteins are involved in every biological process. Scientific research against any known disease has one or more protein as final target. If any living being can be considered as an envelope protecting its most important treasure, the genetic information, then proteins must be thought as both results of the information and tools for protecting, conserving and passing it on. In this point of view, “proteomics” is not just that somewhat mysterious core facility down the building, where “upstair” scientists bring their samples just to know “who's in that 2D gel spot”. Proteomics is putting every protein in place in its correct biochemical contest, assessing its identity, its modifications map, its degree of over or underexpression, its folding and its interactions with other proteins. In this extremely ambitious project, mass spectrometry plays a crucial role, because the development of electrospray and MALDI ion sources (John B. Fenn and Koichi Tanaka, Nobel laureates in 2002) opened a brand new world, where even molecular “elephants” can be transferred in the gas phase, opening unique and almost endless possibilities for protein analysis. The aim of this special issue, composed of eight reviews, is to give the reader an introductory but exhaustive overview of the analytical techniques used to purify, identify, quantify and characterize proteins, keeping the main focus on mass spectrometry. The first two articles have been written by William Ward and Gert Van Den Berg, acknowledged experts on protein purification and 2D gel electrophoresis, respectively. These two steps are the foundation of the MS-based protein research, because they allow a protein to be purified from its biological environment, an extremely complex analytical matrix. The third review describes how a protein can be identified, through a “bottom-up” process and the extensive use of bioinformatics tools, whose key role in proteomics is to extract significant results from the enormous amount of data generated by mass spectrometry. This last topic is covered by a notable review of Michael Goshe. Post translational modifications of proteins, such as phosphorylations and glycosilations, play a crucial role in their biological activity and are one of the most addressed issues in proteomics. In his review, Willy Morelle exhaustively describes the most important techniques for PTMs analysis. The fifth article, written by Michael Goshe again, brightly describes an array of relatively new tools for quantitative proteomics, based on both label and label-free methods. The last two papers deal with an area of protein science that is not usually included (unfortunately) in classical MS-based “proteomics” reviews. These two articles focus on a frontier area of mass spectrometry: the study of protein structure, folding and function. Such an exciting field of research is greatly discussed by Lars Konermann and John Engen. The Authors and the Editor sincerely hope that this special issue will represent an outline of the state of the art of several fields of proteomic sciences, possibly representing an introductory approach for those analytical scientist not (yet?) directly involved in protein research. Finally, as Guest Editor of this special issue I would like to express my gratitude to the Authors for their excellent work and to the Publishers for giving me the opportunity to work with such a brilliant group of scientists.

The science, art, and practice of protein purification have been with us for more than a century, yet, in many respects, the field is only now evolving past its adolescent roots. New methods are replacing old methods at such a dizzying pace, that even life-long experts in protein purification cannot keep up. In this article, we present many state-of-the-art protein purification techniques without totally ignoring the past. Our goal is to enable those relatively new to the field of protein purification to choose the best methods to solve their own purification problems. Each method we describe has been used and validated in our own research. We describe these methods, pointing out advantages, disadvantages, and limitations with practical examples rather than with complex, theoretical equations. This paper covers methods of extraction, clarification, batch purification, low pressure column chromatography, HPLC, and electrophoresis as applied to both genetically engineered, recombinant proteins and proteins isolated from natural sources. The relatively new methods of three-phase partitioning, hydrophobic charge induction chromatography, immobilized metal affinity chromatography, and perfusion chromatography are featured.

Two-dimensional electrophoresis has, for many years, been the primary workhorse for performing functional proteomics, the large-scale analysis of protein expression differences. Despite its merits, limitations inherent to this technology have been recognized for a long time, ranging from its gel-to-gel variability to its inability to represent several classes of proteins. Recently, however, technical advances in two-dimensional electrophoresis have alleviated several of these drawbacks. Fractionation approaches prior to two-dimensional electrophoresis, e.g. by chromatography, organelle fractionation or Equalizer Beads technology, have increased the number of visible proteins. Fluorescent two-dimensional difference gel electrophoresis has boosted the quantitative aspects of two-dimensional electrophoresis. New protein stains have also enabled the analysis of post-translationally modified proteins. As a result, two-dimensional electrophoresis has been thoroughly modernized, enabling it to remain the preferred method for protein expression analysis in a large number of laboratories. In this review we will give an overview of these technological advances.

In this work, the “bottom-up” protein identification process is described, from sample digestion to the final database search result. Both MALDI and LC-MS approaches to the identification issue are illustrated. The state-of-the-art of these technique is outlined along with several recent applications, such as MSn data acquisition and chemical derivatization of peptides.

Central to the successful implementation of a proteomics pipeline are appropriate bioinformatics tools to provide a level of automation and standardization to the qualitative identification of proteins, quantitation of protein changes, data capture and storage, and integration with other data platforms. Many of these efforts are in various stages of maturity; however, the identification, or recognition, of peptides/proteins from mass spectrometry data, arguably the most developed area, continues to remain challenging due to the ever increasing size of proteomic datasets. Confident peptide and protein identification, including assignment of any post-translational modifications, is a necessary prerequisite for any proteomic study aimed at elucidating biological and physiological responses. This review includes discussions of bioinformatic approaches for the qualitative identification of peptides/proteins from mass spectrometry data as well as the software tools and the analytical considerations required for analysis. Issues related to placing a proteomics dataset in a larger biological context which includes splice variants, variations in databases, and neglected proteomes are also discussed.

The determination of post-translational modifications is one of the main challenges in proteomics research. Mass spectrometry is a powerful tool for the structural characterization of proteins and different mass spectrometric techniques for the analysis of post-translational modifications of individual proteins or protein populations have been developed. This review describes the most recent advances in mass spectrometry-based approaches for the detection and determination of post-translational modifications, with an emphasis on glycosylation and phosphorylation.

Mass spectrometry is an extremely versatile analytical technique that is capable of characterizing proteins at various levels of biochemical sophistication from recognition of protein components and their modifications to their quantification within a sample. With the development of electrospray ionization and matrix-assisted laser desorption ionization, the last decade of protein analysis using mass spectrometry has fully established the field of proteomics within the life sciences and a major player in the systems biology paradigm. The diversity of proteins and their multi-facetted functions are indicative of the numerous mass spectrometry methods that are used in quantitative proteomic analysis. In this review, the various techniques developed to quantify protein abundance by mass spectrometry are presented in terms of those associated with both stable isotope coding and label-free strategies. The implementation of these methods to the quantitative mass spectrometry analysis from “proof-of-concept” to those that tackle investigations of protein expression and those of protein function mediated by post-translation modifications are also discussed.

Electrospray ionization (ESI) mass spectrometry (MS) has become an indispensable tool for studies on protein structure, folding, dynamics, and interactions. The ESI process generates intact and multiply protonated ions from proteins in solution. The charge state distribution of these ions provides a highly sensitive probe for the overall compactness of a protein in solution. Unfolded conformers lead to the formation of higher charge states than natively folded proteins. Due to its very gentle nature, ESI allows the transfer of intact noncovalent assemblies (protein-ligand and protein-protein complexes) into the gas phase. Thus, ESI-MS is ideally suited for monitoring coupled folding/binding events. The remarkable selectivity of this technique facilitates the observation of co-existing conformers and binding states. This review discusses mechanistic aspects of the ionization process, as well as selected examples that illustrate the use of ESI-MS for monitoring protein folding and assembly reactions. The combination of ESI-MS with on-line mixing techniques can provide mechanistic insights into processes occurring on very rapid time scales. We also address the interesting question whether biomolecular structures in the gas phase resemble those in solution. Experimental approaches involving hydrogen exchange and covalent labeling techniques are covered in an accompanying article.

Mass spectrometry can be used to obtain information about all levels of protein structure. To gain access to the conformation information found in the tertiary and quaternary structure, various labeling methods have been developed. These methods convert structural information into mass differences that can be observed with high-resolution protein/ peptide mass spectrometry. Three methods are reviewed here: hydrogen/deuterium exchange, covalent modification (also called chemical modification) and hydroxyl radical footprinting. The general implementation of these methods is described and comparisons are made between the methods.