Zon Weng Lai

Emerging Techniques in Proteomics for Probing Nano–Bio Interactions

Nanoengineered particles that can facilitate drug formulation and improve specificity of delivery afford exciting opportunities for improved lesion-specific therapy. Understanding and controlling the nano-bio interactions of these materials is central to future developments in this area. Mass-spectrometry-based proteomics techniques, in conjunction with other emerging technologies, are enabling novel insights into the modulation of particle surfaces by biological fluids (formation of the protein corona) and subsequent particle-induced cellular responses. In this Perspective, we summarize important recent developments using proteomics-based techniques to understand nano-bio interactions and discuss the impact of such knowledge on improving particle design.

Protein amino-terminal modifications and proteomic approaches for N-terminal profiling

Amino-/N-terminal processing is a crucial post-translational modification affecting almost all proteins. In addition to altering the chemical properties of the N-terminus, these modifications affect protein activation, conversion, and degradation, which subsequently lead to diversified biological functions. The study of N-terminal modifications is of increasing interest; especially since modifications such as proteolytic truncation or pyroglutamate formation have been linked to disease processes. During the past decade, mass spectrometry has played an important role in facilitating the investigation of N-terminal modifications. Continuous progress is being made in the development and application of robust methods for the dedicated analysis of native and modified protein N-termini in a proteome-wide manner. Here we highlight recent progress in our understanding of protein N-terminal biology as well as outlining present enrichment strategies for mass spectrometry-based studies of protein N-termini.

Particles on the move: intracellular trafficking and asymmetric mitotic partitioning of nanoporous polymer particles.

Nanoporous polymer particles (NPPs) prepared by mesoporous silica templating show promise as a new class of versatile drug/gene delivery vehicles owning to their high payload capacity, functionality, and responsiveness. Understanding the cellular dynamics of such particles, including uptake, intracellular trafficking, and distribution, is an important requirement for their development as therapeutic carriers. Herein, we examine the spatiotemporal map of the cellular processing of submicrometer-sized disulfide-bonded poly(methacrylic acid) (PMASH) NPPs in HeLa cells using both flow cytometry and fluorescence microscopy. The data show that the PMASH NPPs are transported from the early endosomes to the lysosomes within a few minutes. Upon cell division, the lysosome-enclosed PMASH NPPs are distributed asymmetrically between two daughter cells. Statistical analysis of cells during cytokinesis suggests that partitioning of particles is biased with an average segregation deviation of 60%. Further, two-dimensional difference gel electrophoresis (2D-DIGE) analysis reveals that 127 out of 3059 identified spots are differentially regulated upon exposure to the PMASH NPPs. Pathway analysis of the proteomics data suggests that ubiquitylation, a reversible modification of cellular proteins with ubiquitin, plays a central role in overall cellular responses to the particles. These results provide important insights into the cellular dynamics and heterogeneity of NPPs, as well as the mechanisms that regulate the motility of these particles within cells, all of which have important implications for drug susceptibility characteristics in cancer cells using particle-based carriers.

The emerging role of the peptidome in biomarker discovery and degradome profiling.

Abstract The peptidome represents the array of endogenous peptides that are present in both the intracellular and extracellular space of the body. Peptides are constantly generated in vivo by active synthesis, and by proteolytic processing of larger precursor proteins, often yielding protein fragments that mediate a variety of physiological functions. Given that aberrant proteolysis is a hallmark of various pathological diseases, many studies have now turned to the peptidome. Differential regulation of endogenous peptides may play a role in many pathological conditions. Mass spectrometry (MS) -based investigation of peptides in a system-wide manner is currently facilitating the identification of potential biomarkers. Furthermore, peptidomic approaches have provided major contributions to the identification of protease-substrate relationships; representing one of the major challenges in understanding and therapeutically exploiting protease function in health and disease. As such, degradomic studies looking for cleavage products via peptidomics in particular, have warranted a significant research interest in recent years. Given that substantial studies are accumulating in the field of peptidomics, this review highlights recent advances of MS-based peptidomic strategies in facilitating the identification of potential peptides as novel clinical markers and protease-substrate profiling.

Quantitative proteomic analysis of formalin–fixed, paraffin–embedded clear cell renal cell carcinoma tissue using stable isotopic dimethylation of primary amines

BackgroundFormalin-fixed, paraffin-embedded (FFPE) tissues represent the most abundant resource of archived human specimens in pathology. Such tissue specimens are emerging as a highly valuable resource for translational proteomic studies. In quantitative proteomic analysis, reductive di-methylation of primary amines using stable isotopic formaldehyde variants is increasingly used due to its robustness and cost-effectiveness.ResultsIn the present study we show for the first time that isotopic amine dimethylation can be used in a straightforward manner for the quantitative proteomic analysis of FFPE specimens without interference from formalin employed in the FFPE process. Isotopic amine dimethylation of FFPE specimens showed equal labeling efficiency as for cryopreserved specimens. For both FFPE and cryopreserved specimens, differential labeling of identical samples yielded highly similar ratio distributions within the expected range for dimethyl labeling. In an initial application, we profiled proteome changes in clear cell renal cell carcinoma (ccRCC) FFPE tissue specimens compared to adjacent non–malignant renal tissue. Our findings highlight increased levels of glyocolytic enzymes, annexins as well as ribosomal and proteasomal proteins.ConclusionOur study establishes isotopic amine dimethylation as a versatile tool for quantitative proteomic analysis of FFPE specimens and underlines proteome alterations in ccRCC.

Carboxyterminal protein processing in health and disease: key actors and emerging technologies.

Carboxypeptidases are important mediators of cellular behavior. Through C-terminal truncations, they alter protein functionality and participate in proteome turnover. Similarly, carboxypeptidases shape the human peptidome by targeting neuroendocrine and vasoactive peptides, thereby regulating signaling pathways in the nervous and cardiovascular systems as well as in embryonic development. Carboxypeptidases are widely connected to various pathological processes such as carcinogenesis and neurodegenerative and cardiovascular diseases. The repertoire of carboxypeptidase in vivo substrates still remains poorly defined, largely due to the lack of suitable experimental approaches. Understanding the precise role of carboxypeptidases is pivotal in the future development of diagnostic/prognostic markers in such diseases. To date, very little attention has been paid to the implication of carboxypeptidases in shaping the proteome as well as the peptidome. This review focuses on the patho-physiological function of carboxypeptidases and highlights the approaches by which proteomics-based technologies can be applied to characterize carboxypeptidases and to quantify the differential regulation of proteins by carboxypeptidases in a proteome-wide manner.

Enrichment of protein N-termini by charge reversal of internal peptides

Protein N‐termini provide useful information for the understanding of posttranslational processing of proteins. The majority of proteins undergo N‐terminal processing, such as proteolytic truncation or modifications like acetylation. Multiple methods currently exist for the enrichment of N‐terminal peptides for proteomic analyses. Here, we report a novel, simple, and straightforward N‐terminomic strategy, based on charge reversal of internal peptides followed by their removal through strong cation exchange chromatography. Our initial proof‐of‐concept study shows the feasibility of this technique, yielding over 3000 identifications of protein N‐termini. We further show the application of this strategy in investigating the N‐terminome of mouse embryonic fibroblasts cells deficient for both cathepsin B and L in comparison to wild type) control cells. Finally, we demonstrate that this workflow can be used in combination with a gel‐based strategy, allowing preseparation of proteins and thus providing an estimate of the molecular weight of the identified cleavage products.

Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini

Dysregulated proteolysis represents a hallmark of numerous diseases. In recent years, increasing number of studies has begun looking at the protein termini in hope to unveil the physiological and pathological functions of proteases in clinical research. However, the availability of cryopreserved tissue specimens is often limited. Alternatively, formalin-fixed, paraffin-embedded (FFPE) tissues offer an invaluable resource for clinical research. Pathologically relevant tissues are often stored as FFPE, which represent the most abundant resource of archived human specimens. In this study, we established a robust workflow to investigate native and protease-generated protein N termini from FFPE specimens. We demonstrate comparable N-terminomes of cryopreserved and formalin-fixed tissue, thereby showing that formalin fixation/paraffin embedment does not proteolytically damage proteins. Accordingly, FFPE specimens are fully amenable to N-terminal analysis. Moreover, we demonstrate feasibility of FFPE-degradomics in a quantitative N-terminomic study of FFPE liver specimens from cathepsin L deficient or wild-type mice. Using a machine learning approach in combination with the previously determined cathepsin L specificity, we successfully identify a number of potential cathepsin L cleavage sites. Our study establishes FFPE specimens as a valuable alternative to cryopreserved tissues for degradomic studies.

Comprehensive Proteomic Analysis of Nitrogen-Starved Mycobacterium smegmatis Δpup Reveals the Impact of Pupylation on Nitrogen Stress Response

Pupylation is a bacterial ubiquitin-like protein modification pathway, which results in the attachment of the small protein Pup to specific lysine residues of cellular targets. Pup was shown to serve as a degradation signal, directing proteins toward the bacterial proteasome for turnover. Recently, it was hypothesized that pupylation and proteasomal protein degradation support the survival of Mycobacterium smegmatis (Msm) during nitrogen starvation by supplying recycled amino acids. In the present study we generated a Pup deletion strain to investigate the influence of pupylation on Msm proteome in the absence of nitrogen sources. Quantitative proteomic analyses revealed a relatively low impact of Pup on MsmΔpup proteome immediately after exposure to growth medium lacking nitrogen. Less than 5.4% of the proteins displayed altered cellular levels when compared to Msm wild type. In contrast, post 24 h of nitrogen starvation 501 proteins (41% of the total quantified proteome) of Msm pup deletion strain showed significant changes in abundance. Noteworthy, important players involved in nitrogen assimilation were significantly affected in MsmΔpup. Furthermore, we quantified pupylated proteins of nitrogen-starved Msm to gain more detailed insights in the role of pupylation in surviving and overcoming the lack of nitrogen.

FoxO1 Is Required for Most of the Metabolic and Hormonal Perturbations Produced by Hepatic Insulin Receptor Deletion in Male Mice

Insulin coordinates the complex response to feeding, affecting numerous metabolic and hormonal pathways. Forkhead box protein O1 (FoxO1) is one of several signaling molecules downstream of insulin; FoxO1 drives gluconeogenesis and is suppressed by insulin. To determine the role of FoxO1 in mediating other actions of insulin, we studied mice with hepatic deletion of the insulin receptor, FoxO1, or both. We found that mice with deletion of the insulin receptor alone showed not only hyperglycemia but also a 70% decrease in plasma insulin-like growth factor 1 and delayed growth during the first 2 months of life, a 24-fold increase in the soluble leptin receptor and a 19-fold increase in plasma leptin levels. Deletion of the insulin receptor also produced derangements in fatty acid metabolism, with a decrease in the expression of the lipogenic enzymes, hepatic diglycerides, and plasma triglycerides; in parallel, it increased expression of the fatty acid oxidation enzymes. Mice with deletion of both insulin receptor and FoxO1 showed a much more modest phenotype, with normal or near-normal glucose levels, growth, leptin levels, hepatic diglycerides, and fatty acid oxidation gene expression; however, lipogenic gene expression remained low. Taken together, these data reveal the pervasive role of FoxO1 in mediating the effects of insulin on not only glucose metabolism but also other hormonal signaling pathways and even some aspects of lipid metabolism.