Current Proteomics - Volume 8, Issue 1, 2011

This special edition on Quantitative Mass Spectrometry in Proteomics focuses on some of the leading technologies that are currently used in state-of-the-art laboratories. In recent years, mass spectrometry-based proteomics has come a long way and seen a tremendous advancement to the point where quantitative measurements are no longer a distant goal, but have become routine to some of the leading labs. However, this is not a simple achievement, since it requires an interdisciplinary effort, starting from experimental design, to adequate sample preparation, to state-of-the-art technology, to bioinformatics and statistical analyses. The advancements and possibilities in quantitative proteomics have, in turn, changed the way, biological research can be done. Rather than focusing on a single protein, more ambitious goals are pursued and entire networks of proteins or protein complexes are interrogated in current proteomics experiments. While there are other techniques available, this special edition focuses on metabolically, chemically or enzymatically introduced mass tags and the quantitative study of protein-ligand interactions. The paper by Graham et al. introduces the commonly used stable isotope labeling by amino acids in cell culture (SILAC) technology to biologists and covers a broad range of applications, bioinformatics tools and potential problems that should be considered when using this technology. Noirel et al. introduce the chemical labeling technique iTRAQ. In a meta-analysis of the the current iTRAQ literature, they give advice to researchers on experimental design, biological and technical replicates and data analysis tools. This is followed by a paper by Boja that specifically introduces the iTRAQ technology with high collision dissociation that has become available in present-day Orbitrap technologies. The review by Hajkova et al. covers the recent developments of the proteolytic 18O labeling technique to improve the reliablity of the label, the use of computational tools to quantify peptide and protein ratios, and a new strategy to compare a large number of samples. Finally, Sharon and Robinson describe how they use mass spectrometry to determine the composition, stoichiometry, subunit interactions, and architectural organization of non-covalent protein complexes. This issue is aimed at both mass spectrometry practitioners who want to familiarize themselves with the current state-of-theart as well as biologists who contemplate the pros and cons of the current proteomics technologies. We certainly hope that it will stimulate further research in the exciting area of quantitative mass spectrometry.

Since its introduction in 2002 ‘stable isotope labeling by amino acids in cell culture’ (SILAC) has become a major tool for quantitative proteomics. Stable isotopes are incorporated into proteins by introduction of labeled amino acids in growth medium. This methodology's compatibility with virtually all cell culture conditions, ease of implementation into existing workflows and the high quality quantitative data that can be obtained have made it the quantitative method of choice for many laboratories. Although originally used for mammalian cell cultures, it has been adapted to a number of organisms including yeast, bacteria, plants and higher organisms. This review will look at the use of SILAC as a key tool in quantitative biology. The purpose of this review is to furnish the reader with a basic understanding of the fundamental principles of SILAC including its implementation, application, bioinformatic tools for its analyses and potential problems that one should be mindful of.

Isobaric tags for absolute and relative quantification (iTRAQ) provide the quantitative proteomics community with an easy-to-use tool to examine proteome changes. Five years after the launch of the technique, has the community become familiar with iTRAQ? In this review, we propose to answer this question by looking at two of its facets. On the one hand, we enquire whether iTRAQ is used ‘optimally’; in other words, whether the community is keeping up with the methods that have been devised and the requirements that have become recognised, in quantitative proteomics. On the other hand, there is the question of whether an iTRAQ paper is written in such a way as to allow other researchers to challenge or improve the results. To tackle these two problems, we have reviewed the iTRAQ literature and gathered data about the methods; in this study, we discuss the experimental designs, the use of biological and technical replicates, the confirmation through additional experiments, the strategies to identify differential expression and supplementary information. Our conclusions aim at giving recommendations in light of our statistics.

Multiplexed stable isotope reagents such as iTRAQ and Tandem Mass Tag (TMT) designed for MS/MS-based quantitation of peptides rely on accurate and robust detection of low mass fragments for peptide precursor ions. In the past, such analyses depended upon mass spectrometers capable of producing the so-called “triple-quadrupole-like” fragmentation including triple quadrupole mass spectrometers (TQMS) and quadrupole time-of-flight (Q-TOF) instruments. The “one-third rule” on an ion trap (IT) instrument precluded the use of these reagents on this widely available instrument platform until the invention of Pulsed Q Dissociation (PQD), which in itself manifests problems in generating sufficient reporter ion intensities for accurate quantitation. The introduction of high energy collisional-activated dissociation (HCD) on LTQ-Orbitrap XL and LTQ-Orbitrap Velos platforms has opened up great opportunities for accurate quantitative analysis of proteins and their post-translational modifications (PTMs) using chemical tagging by generating “triple- quadrupole-like” fragmentation mainly in the low mass range in MS/MS mode.

The proteolytic 18O labeling method determines the relative ratios of individual proteins between two samples. This technique utilizes a protease and water (H216O and H218O) to produce labeled peptides; peptides in one sample incorporate 16O by labeling in H216O, and the other sample incorporates 18O by labeling in H218O. Both samples are mixed in a 1:1 ratio and subjected to mass spectrometric analysis to identify and quantify the proteins from which the peptides originated. Technical issues in sample preparation and data processing prevented this method from becoming widely accepted in the field of quantitative proteomics, however these problems have been resolved and the technique is now rapidly gaining popularity. This review focuses on the recent technological developments that have improved the reliability and practicality of proteolytic 18O labeling, including improved peptide labeling techniques, techniques to prevent proteasecatalyzed 18O to 16O back exchange reaction, mass spectrometry platforms suitable for this technique, computational tools for the calculation of 16O/18O-peptide ratios, and a new strategy that allows to compare a large number of samples.

Mass spectrometry has become a powerful tool for determining the composition, stoichiometry, subunit interactions, and architectural organization of non-covalent protein complexes. The vast majority of assemblies studied so far by this approach are those that contain a sufficient amount of electrostatic interactions and hydrogen bonds that can survive the transition from solution to the gas-phase and maintain the structural features of the vaporized ions. An intriguing question that naturally arises is whether mass spectrometry can also be harnessed for the study of molecular systems dominated by non-polar interactions. Here we address this issue and discuss the fate of hydrophobic complexes in the absence of bulk water molecules. We emphasize the progress that has been accomplished in this field that is moving towards the analysis of larger and more complex hydrophobic systems. We attribute this advance to recent developments of mass spectrometry and its application to non-covalent complexes in general, and to the understanding of experimental and biochemical conditions for the preservation of hydrophobic interactions in particular. Furthermore, we discuss the ability of mass spectrometry to serve as a quantitative tool for assessing the strength, binding energies, and stoichiometries of hydrophobic interactions. Overall, we aim to stimulate research in this area and to establish mass spectrometry as a tool for analyzing hydrophobic interactions within complex biological systems.

Organelles and compartments are distinguished by their biological functions, which in turn are reflected by their proteome profiles. Prerequisites for the organization of cellular compartments are mechanisms that help to generate and maintain their proper composition. The nucleolus is one of the compartments that have been the focus of intense research during the past years. It is now well-established that nucleoli are crucial for a growing number of diverse cellular processes. Moreover, it became clear that the functional organization of nucleoli is important for the survival and growth of cells under normal, stress and disease conditions. Recent research provided us with information on the nucleolar proteome under different physiological conditions. These studies demonstrated that multiple chaperone families, cochaperones and other folding factors are present in nucleoli. The exact functions chaperones have in the nucleolus are currently not understood; however, data provided by nucleolar proteomics put us in the position to study novel aspects of chaperone biology. These include the specific roles of chaperones in the nucleolus and the mechanisms that regulate their transport in and out of nucleoli. Our review summarizes the present knowledge of chaperones and their co-factors in the nucleolus as it is emerging from proteomics and other studies. Based on this information, we speculate on the biological relevance of chaperones in the nucleolus and the cellular pathways that promote their targeting to this subnuclear compartment. We conclude by highlighting the open questions that will have to be addressed in future studies.