A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry

The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.     

Vectorial proteomics

Vectorial proteomics is a methodology for the differential identification and characterization of proteins and their domains exposed to the opposite sides of biological membranes. Proteomics of membrane vesicles from defined isolated membranes automatically determine cellular localization of the identified proteins and reduce complexity of protein characterizations. The enzymatic shaving of naturally-oriented, or specifically-inverted sealed membrane vesicles, release the surface-exposed peptides from membrane proteins. These soluble peptides are amenable to various chromatographic separations and to sequencing by mass spectrometry, which provides information on the topology of membrane proteins and on their posttranslational modifications. The membrane shaving techniques have made a breakthrough in the identification of in vivo protein phosphorylation sites in membrane proteins form plant photosynthetic and plasma membranes, and from caveolae membrane vesicles of human fat cells. This approach has also allowed investigation of dynamics for in vivo protein phosphorylation in membranes from cells exposed to different conditions. Vectorial proteomics of membrane vesicles with retained peripheral proteins identify extrinsic proteins associated with distinct membrane surfaces, as well as a variety of posttranslational modifications in these proteins. The rapid integration of versatile vectorial proteomics techniques in the functional characterization of biological membranes is anticipated to bring significant insights in cell biology.     

Immobilized metal affinity chromatography optimized for the analysis of extracellular phosphorylation

Phosphorylation is the most widely studied posttranslational modification. Its role within the cell has been the focus of numerous large‐scale studies. Recently there is growing evidence on the biological significance of extracellular phosphorylation. The analysis of these phosphopeptides is complicated by the abundance of glycosylation in the extracellular space, since glycopeptides are also enriched by the methods used for phosphopeptide isolation. Thus, we optimized IMAC for phosphorylation analysis of secreted proteins, specifically in human serum. Selectivity and efficiency of different enrichment conditions used in earlier large‐scale phosphoproteomic studies were evaluated. We found that minimizing hydrophilic interactions in the enrichment allowed selective phosphopeptide isolation. Using a two‐step IMAC enrichment protocol under these conditions led to the identification of ～100 phosphorylation sites from the tryptic digest of as little as 40 μL human serum.     

Identification of Cep169-interacting proteins and the in vivo modification sites of Cep169 via proteomic analysis

Cep169 is a microtubule plus-end tracking and centrosomal protein that interacts with CDK5RAP2. Cep169 is known to regulate microtubule dynamics and stability; however, its other cellular functions remain largely elusive. In this study, we identified novel Cep169-interacting proteins from HeLa cell extracts. Proteomic analysis via LC-MS/MS helped to identify approximately 400 novel Cep169-interacting proteins, including centrosomal proteins, cilium proteins, microtubule-associating proteins, and several E3 ubiquitin ligases. In addition, we identified in vivo posttranslational modification sites of Cep169, namely, 27 phosphorylation sites, five methylation sites, and four ubiquitination sites. Of these, 14 phosphorylated residues corresponding to the consensus Cdk phosphorylation sites may be required for Cdk1-mediated dissociation of Cep169 from the centrosome during mitosis and Cdk regulation during the G1/S phase. Furthermore, siRNA-induced Cep169 depletion was found to inhibit the growth of RPE1 cells. Our findings suggest that Cep169 regulates cell growth by interacting with multiple proteins.     

NO means no and yes: regulation of cell signaling by protein nitrosylation

Protein nitrosylation is emerging as a key mechanism by which nitric oxide regulates cell signaling. Nitrosylation is the binding of a NO group to a metal or thiol (-SH) on a peptide or protein. Like phosphorylation, nitrosylation is a precisely targeted and rapidly reversible posttranslational modification that allows cells to flexibly and specifically respond to changes in their environment. An increasing number of proteins have been identified whose activity is regulated by intracellular nitrosylation. This review focuses on proteins regulated by endogenous nitrosylation, the chemistry underlying nitrosylation, the specificity and reversibility of nitrosylation reactions, methods to detect protein nitrosylation, and the role of coordinated protein nitrosylation/denitrosylation in cell signaling.     

Production of G protein‐coupled receptors in an insect‐based cell‐free system

The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein‐coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell‐free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell‐free system, performed within a short time and in a cost‐effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect‐based cell‐free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell‐free synthesized MOR in comparison to MOR expressed in a human cell line by “one‐point” radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328–2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Phosphosite mapping of P-type plasma membrane H+-ATPase in homologous and heterologous environments

Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H(+)-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any other P-type ATPase. Phosphosites were almost exclusively (9 of 10) in the terminal regulatory domains of the pumps. The AHA2 isoform was subsequently expressed in the yeast Saccharomyces cerevisiae. The plant protein was phosphorylated at multiple sites in yeast, and surprisingly, seven of nine of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H(+)-ATPase to protein kinase action.     

Phosphorylation of serine residue 89 of human adenovirus E1A proteins is responsible for their characteristic electrophoretic mobility shifts, and its mutation affects biological function.

The shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is characteristic of the adenovirus E1A proteins is the result of posttranslational modification. In the present study, we demonstrate that phosphorylation of bacterially produced E1A in higher cell extracts occurs on serine and is responsible for the mobility shift. E1A protein expressed in Saccharomyces cerevisiae also undergoes the mobility shift due to serine phosphorylation. Site-directed mutagenesis was used to identify the serine residue responsible for the mobility shift. Six serine residues were altered to glycine within E1A. Substitution at serine residue 89 was shown to selectively prevent the mobility shift of both the 289R and 243R E1A proteins. We conclude that phosphorylation at serine 89 is the specific modification responsible for the mobility shift of E1A. Moreover, we demonstrate that the Ser-89-to-Gly mutation has no effect on trans activation or complementation of an E1A-deficient adenovirus. In contrast, the mutant protein does significantly reduce both the repression and transformation efficiency of E1A. The five other Ser-to-Gly mutation were also examined for functional effects. None affected trans activation, whereas repression and transformation functions were affected. One mutant affected transformation without affecting repression, suggesting that these functions are to some degree also separable. The relevance of phosphorylation to structure and activity of E1A and other nuclear oncogene proteins is discussed.     

Phosphorylation Increases Persistence Length and End-to-End Distance of a Segment of Tau Protein

Intrinsically disordered regions of proteins, which lack unique tertiary structure under physiological conditions, are enriched in phosphorylation sites and in significant local bias toward the polyproline II conformation. The overrepresented coincidence of this posttranslational regulatory signal and local conformational bias within unstructured regions raises a question: can phosphorylation serve to manipulate the conformational preferences of a disordered protein? In this study, we use time-resolved fluorescence resonance energy transfer and a, to our knowledge, novel data analysis method to directly measure the end-to-end distance distribution of a phosphorylatable peptide derived from the human microtubule associated protein tau. Our results show that phosphorylation at threonine or serine extends the end-to-end distance and increases the effective persistence length of the tested model peptides. Unexpectedly, the extension is independent of salt concentration, suggestive of a nonelectrostatic origin. The phosphorylation extension and stiffening effect provides a peptide-scale physical interpretation for the posttranslational regulation of the highly abundant protein-protein interactions found in disordered proteins, as well as a potential insight into the regulatory mechanism of the tau protein’s microtubule binding activity.     

VDAC proteomics: Post-translation modifications

Voltage-dependent anion channels are abundant mitochondrial outer membrane proteins expressed in three isoforms, VDAC1-3, and are considered as "mitochondrial gatekeepers". Most tissues express all three isoforms. The functions of VDACs are several-fold, ranging from metabolite and energy exchange to apoptosis. Some of these functions depend on or are affected by interaction with other proteins in the cytosol and intermembrane space. Furthermore, the function of VDACs, as well as their interaction with other proteins, is affected by posttranslational modification, mainly phosphorylation. This review summarizes recent findings on posttranslational modification of VDACs and discusses the physiological outcome of these modifications. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.     

Glycosylated and Phosphorylated Proteins—Expression in Yeast and Oocytes of Xenopus: Prospects and Challenges—Relevance to Expression of Thermostable Proteins

Phosphorylation and glycosylation are important posttranslational events in the biosynthesis of proteins. The different degrees of phosphorylation and glycosylation of proteins have been an intriguing phenomenon. Advances in genetic engineering have made it possible to control the degree of glycosylation and phosphorylation of proteins. Structural biology of phosphorylated and glycosylated proteins has been advancing at a much slower pace due to difficulties in using high-resolution NMR studies in solution phase. Major difficulties have arisen from the inherent mobilities of phosphorylated and glycosylated side chains. This paper reviews molecular and structural biology of phosphorylated and glycosylated proteins expressed in eukaryotic expression systems which are especially suited for large-scale production of these proteins. In our laboratory, we have observed that eukaryotic expression systems are particularly suited for the expression of thermostable light-activated proteins, e.g., bacteriorhodopsins and plastocyanins.     

Glycosylated and phosphorylated proteins--expression in yeast and oocytes of Xenopus: prospects and challenges--relevance to expression of thermostable proteins

Phosphorylation and glycosylation are important posttranslational events in the biosynthesis of proteins. The different degrees of phosphorylation and glycosylation of proteins have been an intriguing phenomenon. Advances in genetic engineering have made it possible to control the degree of glycosylation and phosphorylation of proteins. Structural biology of phosphorylated and glycosylated proteins has been advancing at a much slower pace due to difficulties in using high-resolution NMR studies in solution phase. Major difficulties have arisen from the inherent mobilities of phosphorylated and glycosylated side chains. This paper reviews molecular and structural biology of phosphorylated and glycosylated proteins expressed in eukaryotic expression systems which are especially suited for large-scale production of these proteins. In our laboratory, we have observed that eukaryotic expression systems are particularly suited for the expression of thermostable light-activated proteins, e.g., bacteriorhodopsins and plastocyanins.     

Phosphorylation of basic amino acid residues in proteins: important but easily missed

Reversible phosphorylation is the most widespread posttranslational protein modification, playing regulatory role in almost every aspect of cell life. The majority of protein phosphorylation research has been focused on serine, threonine and tyrosine that form acid-stable phosphomonoesters. However, protein histidine, arginine and lysine residues also may undergo phosphorylation to yield acid-labile phosphoramidates, most often remaining undetected in conventional studies of protein phosphorylation. It has become increasingly evident that acid-labile protein phosphorylations play important roles in signal transduction and other regulatory processes. Beside acting as high-energy intermediates in the transfer of the phosphoryl group from donor to acceptor molecules, phosphohistidines have been found so far in histone H4, heterotrimeric G proteins, ion channel KCa3.1, annexin 1, P-selectin and myelin basic protein, as well as in recombinant thymidylate synthase expressed in bacterial cells. Phosphoarginines occur in histone H3, myelin basic protein and capsidic protein VP12 of granulosis virus, whereas phospholysine in histone H1. This overview of the current knowledge on phosphorylation of protein basic amino-acid residues takes into consideration its proved or possible roles in cell functioning. Specific requirements of studies on acid-labile protein phosphorylation are also indicated.     

Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.