Fred Elisma

Large‐scale mapping of human protein–protein interactions by mass spectrometry

Mapping protein–protein interactions is an invaluable tool for understanding protein function. Here, we report the first large‐scale study of protein–protein interactions in human cells using a mass spectrometry‐based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large‐scale immunoprecipitation of Flag‐tagged versions of these proteins followed by LC‐ESI‐MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross‐validated using previously published and predicted human protein interactions. In‐depth mining of the data set shows that it represents a valuable source of novel protein–protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

Lipidomics era: Accomplishments and challenges

Lipid mediators participate in signal transduction pathways, proliferation, apoptosis, and membrane trafficking in the cell. Lipids are highly complex and diverse owing to the various combinations of polar headgroups, fatty acyl chains, and backbone structures. This structural diversity continues to pose a challenge for lipid analysis. Here we review the current state of the art in lipidomics research and discuss the challenges facing this field. The latest technological developments in mass spectrometry, the role of bioinformatics, and the applications of lipidomics in lipid metabolism and cellular physiology and pathology are also discussed.

The Tale of Two Domains Proteomics and Genomics Analysis of SMYD2, A New Histone Methyltransferase

Very little is known about SET- and MYND-containing protein 2 (SMYD2), a member of the SMYD protein family. However, the interest in better understanding the roles of SMYD2 has grown because of recent reports indicating that SMYD2 methylates p53 and histone H3. In this study, we present a combined proteomics and genomics study of SMYD2 designed to elucidate its molecular roles. We report the cytosolic and nuclear interactome of SMYD2 using a combination of immunoprecipitation coupled with high throughput MS, chromatin immunoprecipitation coupled with high throughput MS, and co-immunoprecipitation methods. In particular, we report that SMYD2 interacted with HSP90α independently of the SET and MYND domains, with EBP41L3 through the MYND domain, and with p53 through the SET domain. We demonstrated that the interaction of SMYD2 with HSP90α enhances SMYD2 histone methyltransferase activity and specificity for histone H3 at lysine 4 (H3K4) in vitro. Interestingly histone H3K36 methyltransferase activity was independent of its interaction with HSP90α similar to LSD1 dependence on the androgen receptor. We also showed that the SET domain is required for the methylation at H3K4. We demonstrated using a modified chromatin immunoprecipitation protocol that the SMYD2 gain of function leads to an increase in H3K4 methylation in vivo, whereas no observable levels of H3K36 were detected. We also report that the SMYD2 gain of function was correlated with the up-regulation of 37 and down-regulation of four genes, the majority of which are involved in the cell cycle, chromatin remodeling, and transcriptional regulation. TACC2 is one of the genes up-regulated as a result of SMYD2 gain of function. Up-regulation of TACC2 by SMYD2 occurred as a result of SMYD2 binding to the TACC2 promoter where it methylates H3K4. Furthermore the combination of the SMYD2 interactome with the gene expression data suggests that some of the genes regulated by SMYD2 are closely associated with SMYD2-interacting proteins.

Proteomic analyses of the SMYD family interactomes identify HSP90 as a novel target for SMYD2

The SMYD (SET and MYND domain) family of lysine methyltransferases (KMTs) plays pivotal roles in various cellular processes, including gene expression regulation and DNA damage response. Initially identified as genuine histone methyltransferases, specific members of this family have recently been shown to methylate non-histone proteins such as p53, VEGFR, and the retinoblastoma tumor suppressor (pRb). To gain further functional insights into this family of KMTs, we generated the protein interaction network for three different human SMYD proteins (SMYD2, SMYD3, and SMYD5). Characterization of each SMYD protein network revealed that they associate with both shared and unique sets of proteins. Among those, we found that HSP90 and several of its co-chaperones interact specifically with the tetratrico peptide repeat (TPR)-containing SMYD2 and SMYD3. Moreover, using proteomic and biochemical techniques, we provide evidence that SMYD2 methylates K209 and K615 on HSP90 nucleotide-binding and dimerization domains, respectively. In addition, we found that each methylation site displays unique reactivity in regard to the presence of HSP90 co-chaperones, pH, and demethylation by the lysine amine oxidase LSD1, suggesting that alternative mechanisms control HSP90 methylation by SMYD2. Altogether, this study highlights the ability of SMYD proteins to form unique protein complexes that may underlie their various biological functions and the SMYD2-mediated methylation of the key molecular chaperone HSP90.

Rare Cell Proteomic Reactor Applied to Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of Human Embryonic Stem Cell Differentiation

The molecular basis governing the differentiation of human embryonic stem cells (hESCs) remains largely unknown. Systems-level analysis by proteomics provides a unique approach to tackle this question. However, the requirement of a large number of cells for proteomics analysis (i.e. 106–107 cells) makes this assay challenging, especially for the study of rare events during hESCs lineage specification. Here, a fully integrated proteomics sample processing and analysis platform, termed rare cell proteomic reactor (RCPR), was developed for large scale quantitative proteomics analysis of hESCs with ∼50,000 cells. hESCs were completely extracted by a defined lysis buffer, and all of the proteomics sample processing procedures, including protein preconcentration, reduction, alkylation, and digestion, were integrated into one single capillary column with a strong cation exchange monolith matrix. Furthermore, on-line two-dimensional LC-MS/MS analysis was performed directly using RCPR as the first dimension strong cation exchange column. 2,281 unique proteins were identified on this system using only 50,000 hESCs. For stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative study, a ready-to-use and chemically defined medium and an in situ differentiation procedure were developed for complete SILAC labeling of hESCs with well characterized self-renewal and differentiation properties. Mesoderm-enriched differentiation was studied by RCPR using 50,000 hESCs, and 1,086 proteins were quantified with a minimum of two peptides per protein. Of these, 56 proteins exhibited significant changes during mesoderm-enriched differentiation, and eight proteins were demonstrated for the first time to be overexpressed during early mesoderm development. This work provides a new platform for the study of rare cells and in particular for further elucidating proteins that govern the mesoderm lineage specification of human pluripotent stem cells.

Technological developments in lipidomics

Lipid analysis is a well-established field of research that focuses on one lipid or a few lipids. The recent developments in mass spectrometry technologies have enabled more comprehensive studies to be performed on lipids present in a sample. The move towards extensive lipid research has led to the coining of the term lipidomics, which is defined as the ensemble of lipids present in a sample. In this review, we will discuss the technical developments in the field of lipidomics and the current limitations of this nascent field.

Analysis of the subcellular phosphoproteome using a novel phosphoproteomic reactor.

Protein phosphorylation is an important post-translational modification involved in the regulation of many cellular processes. Mass spectrometry has been successfully used to identify protein phosphorylation in specific pathways and for global phosphoproteomic analysis. However, phosphoproteomics approaches do not evaluate the subcellular localization of the phosphorylated forms of proteins, which is an important factor for understanding the roles of protein phosphorylation on a global scale. The in-depth mapping of protein phosphorylation at the subcellular level necessitates the development of new methods capable of specifically and efficiently enriching phosphopeptides from highly complex samples. Here, we report a novel microfluidic device called the phosphoproteomic reactor that combines efficient processing of proteins followed by phosphopeptide enrichment by Ti-IMAC. To illustrate the potential of this novel technology, we mapped the phosphoproteins in subcellular organelles of liver cells. Fifteen subcellular fractions from liver cell cultures were processed on the phosphoproteomic reactor in combination with nano-LC-MS/MS analysis. We identified thousands of phosphorylation sites in over 600 phosphoproteins in different organelles using minute amounts of starting material. Overall, this approach provides a new avenue for studying the phosphoproteome of the subcellular organelles.

Discovery of Substrates for a SET Domain Lysine Methyltransferase Predicted by Multistate Computational Protein Design

Characterization of lysine methylation has proven challenging despite its importance in biological processes such as gene transcription, protein turnover, and cytoskeletal organization. In contrast to other key posttranslational modifications, current proteomics techniques have thus far shown limited success at characterizing methyl-lysine residues across the cellular landscape. To complement current biochemical characterization methods, we developed a multistate computational protein design procedure to probe the substrate specificity of the protein lysine methyltransferase SMYD2. Modeling of substrate-bound SMYD2 identified residues important for substrate recognition and predicted amino acids necessary for methylation. Peptide- and protein- based substrate libraries confirmed that SMYD2 activity is dictated by the motif [LFM]-1-K(∗)-[AFYMSHRK]+1-[LYK]+2 around the target lysine K(∗). Comprehensive motif-based searches and mutational analysis further established four additional substrates of SMYD2. Our methodology paves the way to systematically predict and validate posttranslational modification sites while simultaneously pairing them with their associated enzymes.

Identification of Protein-Protein Interactions by Mass Spectrometry Coupled Techniques

The use of mass spectrometry in protein identification has revolutionized the field of proteomics. Coupled to various affinity purification techniques, mass spectrometry is used to identify protein-protein interactions. This chapter looks at the use of these affinity purification techniques in the identification of protein interactions. Various tags are used to purify protein complexes including tandem affinity purification. The FLAG tag is another commonly used tag which is a small tag that tends not to interfere with the protein function. These different affinity purification methods are used to purify proteins that are further identified by either ESI-MS or MALDI-MS.

Phosphoproteome analysis of an early onset mouse model (TgCRND8) of Alzheimer's disease reveals temporal changes in neuronal and glia signaling pathways

Sustained exposure to soluble amyloid β (Aβ42) oligomers is predicted to impair synaptic function in the hippocampal‐entorhinal circuit, signaling synaptic loss and precipitating cognitive impairment in Alzheimers disease. Regional changes in overall patterns of protein phosphorylation are likely crucial to promote transition from a presymptomatic to a symptomatic state in response to accumulating Aβ42. Here, we used unbiased proteomic approaches to compare the phosphoproteome of presymptomatic and symptomatic TgCRND8 mice and identify network disruptions in signaling pathways implicated in the manifestation of behavioral indices of learning and memory impairment. Phosphopeptide enrichment with triple isotopic dimethylation labeling combined with online multidimensional separation and MS was used to profile phosphoproteome changes in 2‐ and 6‐month‐old TgCRND8 mice and congenic littermate controls. We identified 1026 phosphopeptides representing 1168 phosphorylation sites from 476 unique proteins. Of these, 595 phosphopeptides from 293 unique proteins were reliably quantified and 139 phosphopeptides were found to change significantly in the hippocampus of TgCRND8 mice following conversion from a presymptomatic to a symptomatic state.