Armand G. Ngounou Wetie

Investigation of stable and transient protein–protein interactions: Past, present, and future

This article presents an overview of the literature and a review of 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 PTMs within the PPIs is also discussed. We focus on methods to study PPIs and methods of detecting PPIs, with particular emphasis on electrophoresis‐based and MS‐based investigation of PPIs, including specific examples. The validation of PPIs is emphasized and the limitations of the current methods for studying stable and transient PPIs are discussed. Perspectives regarding PPIs, with focus on bioinformatics and transient PPIs are also provided.

Protein–protein interactions: switch from classical methods to proteomics and bioinformatics-based approaches

Following the sequencing of the human genome and many other organisms, research on protein-coding genes and their functions (functional genomics) has intensified. Subsequently, with the observation that proteins are indeed the molecular effectors of most cellular processes, the discipline of proteomics was born. Clearly, proteins do not function as single entities but rather as a dynamic network of team players that have to communicate. Though genetic (yeast two-hybrid Y2H) and biochemical methods (co-immunoprecipitation Co-IP, affinity purification AP) were the methods of choice at the beginning of the study of protein–protein interactions (PPI), in more recent years there has been a shift towards proteomics-based methods and bioinformatics-based approaches. In this review, we first describe in depth PPIs and we make a strong case as to why unraveling the interactome is the next challenge in the field of proteomics. Furthermore, classical methods of investigation of PPIs and structure-based bioinformatics approaches are presented. The greatest emphasis is placed on proteomic methods, especially native techniques that were recently developed and that have been shown to be reliable. Finally, we point out the limitations of these methods and the need to set up a standard for the validation of PPI experiments.

Disulfide proteomics for identification of extracellular or secreted proteins.

The combination of SDS‐PAGE and MS is one of the most powerful and perhaps most frequently used gel‐based proteomics approaches in protein identification. However, one drawback of this method is that separation takes place under denaturing and reducing (R) conditions and as a consequence, all proteins with identical apparent molecular mass (Mr) will run together. Therefore, low‐abundant proteins may not be easily identified. Another way of investigating proteins by proteomics is by analyzing subproteomes from a total proteome such as phosphoproteomics, glycoproteomics, or disulfide proteomics. Here, we took advantage of the property of secreted proteins to form disulfide bridges and investigated disulfide‐linked proteins, using SDS‐PAGE under nonreducing (NR) conditions. We separated sera from normal subjects and from patients with various diseases by SDS‐PAGE (NR) and (R) conditions, followed by LC‐MS/MS analysis. Although we did not see any detectable difference between the sera separated by SDS‐PAGE(R), we could easily identify the disulfide‐linked proteins separated by SDS‐PAGE (NR). LC‐MS/MS analysis of the disulfide‐linked proteins correctly identified haptoglobin (Hp), a disulfide‐linked protein usually found as a heterotetramer or as a disulfide‐linked heteropolymer. Western blotting under NR and R conditions using anti‐Hp antibodies confirmed the LC‐MS/MS experiments and further confirmed that upon reduction, the disulfide‐linked Hp heterotetramers and polymers were no longer disulfide‐linked polymers. These data suggest that simply by separating samples on SDS‐PAGEunder NR conditions, a different, new proteomics subset can be revealed and then identified.

Automated Mass Spectrometry-Based Functional Assay for the Routine Analysis of the Secretome

The secretome represents the set of proteins secreted into the extracellular space of cells. These proteins have been shown to play a major role in cell-cell communication. For example, recent observations revealed the presence of diffusible factors with proliferative properties in the secretome of cancer cells. Thus, a qualitative and quantitative analysis of the secretome could lead to the identification of these factors and consequently to the development of new therapeutic strategies. Here, we provide an automated simple and effective strategy to identify novel targets in the secretome of specifically treated cells using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Furthermore, we explore the supportive role of mass spectrometry (MS) in the development of functional assays of identified secreted target molecules. Simplicity is achieved by growing cells in medium free of serum, which eliminates the need to remove the most abundant serum proteins and at the same time reduces disturbing matrix effects. Upon identification of these factors, their validation and characterization will follow. Moreover, this approach can also lead to the identification of proteins abnormally secreted, shed, or oversecreted by cells as response to a stimulus. Furthermore, we also discuss the problems that one may encounter. Finally, we discuss the broad application of automated MS-based proteomics, particularly in cancer research, highlighting new horizons for the use of MS.

Automatic Determination of Disulfide Bridges in Proteins

Precise determination of disulfide linkages between cysteine (Cys) residues in proteins is essential in the determination of protein structure. Therefore, a reliable automated method for the identification of disulfide bridges can serve as an important tool in the analysis of the tertiary structure of proteins of interest. Here, we describe the current and past methods used to identify disulfide bridges in proteins, with a focus on mass spectrometry (MS)–based methods and a particular emphasis on nanoliquid chromatography–tandem mass spectrometry (nanoLC-MS/MS)–based methods. We also show the development of an easy method based on the separation of disulfide-linked proteins by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under denaturing and nonreducing conditions and selective in-gel digestion of proteins using reducing and nonreducing conditions, followed by analysis of the resulting peptide mixture by nanoACQUITY UPLC coupled to a quadrupole time-of-flight (QTOF) Micro mass spectrometer (nanoLC-MS/MS). Data-dependent analysis (DDA) nanoLC-MS/MS and information-dependent analysis (IDA) nanoLC-MS/MS were used for random and targeted identification of disulfide-linked peptides. Finally, an example of electrospray-MS (ESI-MS) and ESI-MS/MS–based determination of disulfide-linked peptides is shown.

Mass spectrometry investigation of glycosylation on the NXS/T sites in recombinant glycoproteins.

We used a targeted proteomics approach to investigate whether introduction of new N-linked glycosylation sites in a chimeric protein influence the glycosylation of the existing glycosylation sites. To accomplish our goals, we over-expressed and purified a chimeric construct that contained the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 (IgG-Fc-ZP3E7 protein). Immunoglobulin heavy chain (IgG-HC protein) was used as control. We then analyzed the IgG-HC and IgG-Fc-ZP3E7 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by Western blotting (WB). We concluded that in control experiments, the glycosylation site was occupied as expected. However, in the IgG-Fc-ZP3E7 protein, we concluded that only one out of three NXS/T glycosylation sites is occupied by N-linked oligosaccharides. We also concluded that in the IgG-Fc-ZP3E7 protein, upon introduction of additional potential NXS/T glycosylation sites within its sequence, the original NST/S glycosylation site from the Fc region of the IgG-Fc-ZP3E7 protein is no longer glycosylated. The biomedical significance of our findings is discussed.

A Pilot Proteomic Analysis of Salivary Biomarkers in Autism Spectrum Disorder

Autism spectrum disorder (ASD) prevalence is increasing, with current estimates at 1/68–1/50 individuals diagnosed with an ASD. Diagnosis is based on behavioral assessments. Early diagnosis and intervention is known to greatly improve functional outcomes in people with ASD. Diagnosis, treatment monitoring and prognosis of ASD symptoms could be facilitated with biomarkers to complement behavioral assessments. Mass spectrometry (MS) based proteomics may help reveal biomarkers for ASD. In this pilot study, we have analyzed the salivary proteome in individuals with ASD compared to neurotypical control subjects, using MS‐based proteomics. Our goal is to optimize methods for salivary proteomic biomarker discovery and to identify initial putative biomarkers in people with ASDs. The salivary proteome is virtually unstudied in ASD, and saliva could provide an easily accessible biomaterial for analysis. Using nano liquid chromatography‐tandem mass spectrometry, we found statistically significant differences in several salivary proteins, including elevated prolactin‐inducible protein, lactotransferrin, Ig kappa chain C region, Ig gamma‐1 chain C region, Ig lambda‐2 chain C regions, neutrophil elastase, polymeric immunoglobulin receptor and deleted in malignant brain tumors 1. Our results indicate that this is an effective method for identification of salivary protein biomarkers, support the concept that immune system and gastrointestinal disturbances may be present in individuals with ASDs and point toward the need for larger studies in behaviorally‐characterized individuals. Autism Res 2015, 8: 338–350.

Mass spectrometry for the detection of potential psychiatric biomarkers

The search for molecules that can act as potential biomarkers is increasing in the scientific community, including in the field of psychiatry. The field of proteomics is evolving and its indispensability for identifying biomarkers is clear. Among proteomic tools, mass spectrometry is the core technique for qualitative and quantitative identification of protein markers. While significant progress has been made in the understanding of biomarkers for neurodegenerative diseases such as Alzheimer’s disease, multiple sclerosis and Parkinson’s disease, psychiatric disorders have not been as extensively investigated. Recent and successful applications of mass spectrometry-based proteomics in fields such as cardiovascular disease, cancer, infectious diseases and neurodegenerative disorders suggest a similar path for psychiatric disorders. In this brief review, we describe mass spectrometry and its use in psychiatric biomarker research and highlight some of the possible challenges of undertaking this type of work. Further, specific examples of candidate biomarkers are highlighted. A short comparison of proteomic with genomic methods for biomarker discovery research is presented. In summary, mass spectrometry-based techniques may greatly facilitate ongoing efforts to understand molecular mechanisms of psychiatric disorders.

Identification of Post-Translational Modifications by Mass Spectrometry

Proteins are the effector molecules of many cellular and biological processes and are thus very dynamic and flexible. Regulation of protein activity, structure, stability, and turnover is in part controlled by their post-translational modifications (PTMs). Common PTMs of proteins include phosphorylation, glycosylation, methylation, ubiquitination, acetylation, and oxidation. Understanding the biology of protein PTMs can help elucidate the mechanisms of many pathological conditions and provide opportunities for prevention, diagnostics, and treatment of these disorders. Prior to the era of proteomics, it was standard to use chemistry methods for the identification of protein modifications. With advancements in proteomic technologies, mass spectrometry has become the method of choice for the analysis of protein PTMs. In this brief review, we will highlight the biochemistry of PTMs with an emphasis on mass spectrometry.

Applications of Mass Spectrometry in Proteomics

Characterisation of proteins and whole proteomes can provide a foundation to our understanding of physiological and pathological states and biological diseases or disorders. Constant development of more reliable and accurate mass spectrometry (MS) instruments and techniques has allowed for better identification and quantification of the thousands of proteins involved in basic physiological processes. Therefore, MS-based proteomics has been widely applied to the analysis of biological samples and has greatly contributed to our understanding of protein functions, interactions, and dynamics, advancing our knowledge of cellular processes as well as the physiology and pathology of the human body. This review will discuss current proteomic approaches for protein identification and characterisation, including post-translational modification (PTM) analysis and quantitative proteomics as well as investigation of protein–protein interactions (PPIs).