PROTEOMICS Reviews 2013


  • Michael J. Dunn


This issue of PROTEOMICS contains the 2013 collection of review articles covering various aspects of proteomics technologies and their application in the biosciences. We firmly believe that such an annual collection of review articles is an excellent way for both relative newcomers and experts to keep abreast of developments in the field and we hope that you will find this year's collection of review articles interesting and informative.


Membrane proteins play diverse biologically important structural and functional roles including molecular transport, cell communication and signal transduction. As they are implicated in many disease states this group of proteins are major targets for drug discovery. Membrane proteins are usually under-represented in most LC-MS proteomic studies due to their low abundance and poor solubility. To address this problem, various membrane protein enrichment, solubilisation, digestion and fractionation strategies have been developed. The review by Vuckovic and colleagues discusses established and emerging high-throughput gel-free analytical workflows in membrane proteomics, and the inherent advantages, disadvantages and orthogonality of the various approaches.

Reversible phosphorylation, tightly controlled by protein kinases and phosphatases, plays a central role in mediating biological processes, such as protein-protein interactions, sub-cellular translocation, and activation of many cellular enzymes. Current MS-based phosphoproteomic techniques now facilitate the detection and quantification of tens of thousands of phosphorylation sites from a typical biological sample in a single experiment, presenting a major challenge for subsequent functional analysis. In their article, Guo and colleagues provide a state-of-the-art overview of the functional analysis of site-specific phosphorylation and explore new perspectives and outline future challenges.

Chemical cross-linking combined with mass spectrometry for protein structure determination is a striking example of multidisciplinary success. Subra and colleagues review the advances that have been made in this area, including developments in mass spectrometry that provide high mass accuracy measurement, thereby simplifying the identification of cross-linked peptides. The huge amount of data collected has to be processed with dedicated software capable of proposing distance constraints or a structural model of the protein. In addition, new cross-linkers have been designed which facilitate the detection of cross-links, or the interpretation of mass spectrometry data.

Proteolysis is a key step in MS-based proteomic studies, but conventional in-solution digestion is time-consuming and has a low sensitivity. Recently, various immobilized-enzyme reactors have been developed to provide highly efficient proteolysis. The review by Miyazaki and colleagues focuses on this proteolysis step using protease-immobilized reactors and rapid analysis of protein sequences. Analysis of post-translational modifications using this approach is presented as a model application.

Microfabricated bioanalytical platforms have the potential to revolutionize biological analytics. Their popularity is based on several key properties, such as high flexibility of design, low sample consumption, rapid analysis time and minimization of manual handling steps. An optimal totally integrated chip-based microfluidic device would allow rapid automated workflows starting from cell cultivation and ending with MS-based proteome analysis. The consequent reduction in the number of sample handling and transfer steps would improve the reliability, reproducibility and throughput of proteomic investigations. In their review, Hansmeier and colleagues describe recent developments and strategies to enable and integrate proteomic workflows into microfluidic devices.


The development of the HUPO-PSI's standard data formats and MIAPE guidelines facilitate coordination within the scientific community. These standards provide a framework for instrument and software independent exchange and sharing of proteomics data. However, there remains a perception that PSI standards are challenging to implement in routine laboratory pipelines. In their article, Medina-Aunon and colleagues review the tools available for integrating different data standards and building compliant software. These tools are focused on a range of different data types and support different scenarios, intended for software developers or end users, allowing the standards to be used in a straightforward manner.

LC-MS approaches are widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used in protein expression studies. However, for complex biological samples, label-free LC-MS proteomic experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred from one or more measured peptides. In recent years many computational approaches to compute relative protein quantification of label-free LC-MS data have been published. In their review, Webb-Robertson and colleagues examine the most commonly employed quantification approaches to compute relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.


N-linked glycoproteins play important roles in biological processes, including cell-to-cell recognition, growth, differentiation, and programmed cell death. Specific N-linked glycoprotein changes are associated with disease progression and identification of these N-linked glycoproteins has potential for use in disease diagnosis, prognosis, and prediction of treatments. In their article, Tian and Zhang summarize common strategies for N-linked glycoprotein characterization and their application to identification of glycoprotein changes associated with disease states, such as breast cancer, lung cancer, and prostate cancer.

Glycoproteins are found mostly in the secretome, comprising secreted proteins and protein fragments that are released from the cell surface through ectodomain shedding. Secretomes are considered to be a valuable source of therapeutic targets and novel biomarkers. It is therefore not surprising that many existing biomarkers, including biomarkers for breast, ovarian, prostate, and colorectal cancers are glycoproteins. Analysis of secreted glycoproteins thus has the potential to provide information for early disease diagnosis. Furthermore, the glycan/sugar moiety itself can be used as a chemical “handle” for the targeted analysis of secretomes, thereby reducing sample complexity and allowing detection of low abundance proteins in proteomic workflows. The review by Schilling and colleagues focuses on various glycoprotein enrichment strategies that facilitate proteomics-based technologies for the quantitative analysis of secretomes and cell surface proteomes.

Cell Biology

Ubiquitin is a small protein modifier that is covalently attached to the ε-amino group of lysine residues of protein substrates, generally targeting them for degradation. Using specific anti-diglycine antibodies in combination with mass spectrometry, it is now possible to identify more than 10,000 ubiquitylated sites in a single proteomics study. In addition, it is important to unravel the biological relationship between ubiquitylated substrates and the ubiquitin conjugation machinery. In their article, Heck and colleagues discuss the role of affinity purification-MS in characterizing E3 ligase-substrate complexes. Such strategies have also been adapted to screen for binding partners of both deubiquitylating enzymes and ubiquitin-binding domains. The application of MS to study polyubiquitin linkages is an emerging and important area. They also discuss the future of MS-based proteomics in answering important questions with respect to ubiquitylation.

The article by Darie and colleagues reviews recent advances in the analysis of stable and transient protein-protein interactions (PPIs), with a focus on their function within cells, organs and organisms. The significance of post-translational modifications within PPIs is also discussed. Methods of detecting PPIs using approaches based on electrophoresis and MS are discussed. The need for validation of PPIs is emphasized and the limitations of the current methods for studying stable and transient PPIs are discussed.

MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in post-transcriptional regulation of gene expression through repression of mRNA translation and/or degradation of mRNA transcripts. MS-based proteomics has enabled identification of core components of the miRNA processing pathway and the post-translational modifications that are pivotal in miRNA regulatory mechanisms. Quantitative proteomics has been key in identifying miRNA targets. The review by Pandey and colleagues focuses on the role of proteomics and labeling strategies to understand miRNA biology.

Plant Proteomics

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are key regulators of redox homeostasis in living organisms including plants. As control of redox homeostasis plays a central function in plant biology, redox proteomics could help characterizing the potential roles played by ROS/RNS-induced post-translational modification (PTMs) in plant cells. In their review, Tanou and colleagues focus on two PTMs: protein carbonylation (a marker of protein oxidation) and protein S-nitrosylation, both of which having recently emerged as important regulatory mechanisms during numerous fundamental biological processes. They describe recent progress in proteomic analysis of carbonylated and nitrosylated proteins and highlight advances in understanding the physiological basis of these oxy/nitro modifications in plants. The article by Bykova and Rampitsch also focuses on redox control in plants and describes how various proteomic approaches are being used to investigate reversible redox protein modifications in three important cellular processes; oxidative and nitrosative stress, defense against pathogens, cellular redox response and regulation.

Microorganisms, whether they are prokaryotic organisms like bacteria or eukaryotic organisms like fungi, share the need for a secretome adapted to their environment. The review by Girard and colleagues stresses the necessity to fully characterize the composition of the secretome in order to understand the mechanisms responsible for secretome plasticity in this highly diverse group of organisms.

Phosphate is an essential element for plants and as a result crop farming requires the application of large amounts of phosphate fertilizer. However, the fossilized rock deposits used as a commercial source of such fertilizer are a non-renewable resource that will drive plant production costs up as the global demand for food increases. The article by Millar and Alexova focuses on the potential of proteomics to unravel the common and specific biochemical changes that contribute to phosphate use efficiency in cultivars of rice, maize and oilseed rape. These studies reveal a wide scope of species-specific metabolic strategies that lead to changes in root morphology and metabolism, driven by secretion of specific proteins and alteration of energy metabolism, carbon and nitrogen assimilation inside the root cells. Understanding of the mechanisms underlying plant phosphate use efficiency in crops will be critical for the development of sustainable agriculture practices.

Plants require seventeen essential nutrients, including mineral nutrients that are mainly acquired from soils by roots. In natural soils, the availability of most essential mineral nutrients is limited. Developing crops with high nutrient efficiency is essential for sustainable agriculture. In their article, Tian and colleagues describe how proteomic approaches are being used to characterize molecular mechanisms underlying crop adaptation to mineral nutrient deficiency. They discuss the challenge of integrating the data from such studies into system biology approaches for developing crops with high mineral nutrient efficiency.

Zea mays L. is the commonest cereal crop (839 million tons worldwide in 2012) and has consequently attracted considerable academic and commercial research activity. In their article Pechan and colleagues provide a comprehensive review of proteomics studies performed on maize between 1987 and 2012. A broad range of studies is discussed, the results of which are advancing knowledge of diverse aspects of maize biology.

Until recently, large-scale proteomic investigations were only possible for the few model plant species with complete genome sequence information. In contrast, for many other plant species of economic importance, the lack of genomic sequence data has made proteomic analysis challenging. The review by Boutry and Champagne describes how recent technical advances in mass spectrometry and data search programs, and the increasing availability of genomic and cDNA sequences, have facilitated such proteomic studies, making it possible to identify large sets of proteins from non-model plants.

Animal Proteomics

Growth hormone (GH) is a protein secreted by the anterior pituitary and circulates throughout the body to exert important actions on growth and metabolism. It stimulates the secretion of insulin-like growth factor-I (IGF-I) that mediates some of the growth promoting actions of GH. The GH/IGF-I axis has recently been recognized as important in terms of longevity in organisms ranging from C. elegans to mice. In their review, Ding and colleagues summarize proteomic analyses of plasma and white adipose tissue in two mouse models of GH action, i.e. GH transgenic and GHR receptor null mice. The aim of these studies has been to establish novel plasma biomarkers of GH action as a function of age and to determine differences in adipose tissue depots. They have not only confirmed several known physiological actions of GH, but also resulted in novel protein biomarkers and targets that may be indicative of the aging process and/or new functions of GH. These results may generate new directions for GH and/or aging research.

Regeneration is a complex cellular process that results in the formation of new functional tissue rather than scar tissue following injury. Regeneration is a widespread, but not universal, process among metazoans. Planaria, starfish and some worms can regenerate most of their body, whereas many other species can only regenerate parts of specific tissues or fail to accomplish a functional re-growth, as is the case of the central nervous system of mammals. Research in regenerative medicine may enable the regeneration of organs and/or tissues that do not normally undergo this process. Studies of the regeneration process at the molecular level promises to provide answers to why these processes are not readily activated in mammals. Proteomic based approaches are being recognized as extremely useful in the study of regeneration events in a variety of different animal models, tissues and organs. Coelho and colleagues in their review discuss the current status of this field.


Pancreatic cancer has a poor prognosis and is difficult to diagnose and treat. It has become a popular target for proteomic studies, with the promise of new insights into the molecular mechanisms underlying tumorigenesis, improved therapeutic treatment, the identification of cancer-associated protein signatures and signaling events, as well as characterizing interactions between cancer cells and their microenvironment. The review by Pan and colleagues provides an overview of recent tissue proteomic studies of pancreatic cancer.

I hope that you will find the collection of review articles in the PROTEOMICS Reviews 2013 issue to be interesting and stimulating. Please remember that the Proteomics Clinical Applications Reviews 2013 issue is also published this month (issue 1/2, Volume 7). You should certainly not miss this issue in order to keep up to date with exciting recent developments in the field of biomedical proteomics! Finally, we are already planning the next collection of review articles that will be published in February 2014 as the PROTEOMICS Reviews 2014 (issue 4, Volume 14).


Michael J. Dunn