Complex Cell Extracts Research Articles

Bioorthogonal Peptide Enrichment from Complex Samples Using a Rink-Amide-Based Catch-and-Release Strategy.

Uptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of low abundant exogenous antigens from complex cell extracts is difficult. Mass-spectrometry based proteomics - the ideal analysis tool in this case - requires methods to retrieve such molecules with high efficiency and low background. Here, we present a method for the selective and sensitive enrichment of antigenic peptides from APCs using click-antigens; antigenic proteins expressed with azidohomoalanine (Aha) in place of methionine residues. We here describe the capture of such antigens using a new covalent method namely, alkynyl functionalized PEG-based Rink amide resin, that enables capture of click-antigens via copper-catalyzed azide-alkyne [2 + 3] cycloaddition (CuAAC). The covalent nature of the thus formed linkage allows stringent washing to remove a-specific background material, prior to retrieval peptides by acid-mediated release. We successfully identified peptides from a tryptic digest of the full APC proteome containing femtomole amounts of Aha-labelled antigen, making this a promising approach for clean and selective enrichment of rare bioorthogonally modified peptides from complex mixtures.

UHPLC-HRMS-Based Analysis of S-Hydroxymethyl-Glutathione, GSH, and GSSG in Human Cells.

Glutathione (GSH) is one of the main antioxidant molecules present in cells. It harbors a thiol group responsible for sustaining cellular redox homeostasis. This moiety can react with cellular electrophiles such as formaldehyde yielding the compound S-hydroxymethyl-GSH (HSMGSH). HSMGSH is the substrate of the enzyme alcohol dehydrogenase 5 (ADH5) and thus a key intermediate in formaldehyde metabolism. In this work, we describe a method for the chemical synthesis of HSMGSH and a pipeline to identify this compound in complex cell extracts by means of ultra-high-performance liquid chromatography coupled to high-resolution spectrometry (UHPLC-HRMS). This method also allows determining GSH and oxidized disulfide (GSSG) in the same samples, thus providing broad information about formaldehyde-GSH metabolism.

Identification of additive formulations for enhanced shelf‐life and productivity of E. coli cell‐free protein synthesis extracts

Cell‐Free Protein Synthesis (CFPS) is a platform biotechnology that enables a breadth of applications, from expressing traditionally intractable proteins to metabolic engineering and point‐of‐care diagnostics. However, field applications have remained limited due to the poor shelf stability of aqueous E. coli cell‐free extracts, which contain a complex mixture of cellular machinery. Lyophilization of the E. coli extract improves shelf‐life, but remains insufficient for maintaining extract productivity over extended storage periods at room temperature. For many pure protein‐based biologics, a variety of additives have been identified that help to stabilize the protein for lyophilization, transportation, and storage. However, the advances made for pure proteins cannot be directly applied to the complex cell extract, and current investigations into improving cell extract stability via additives has been limited. Thus, we have addressed the cold chain limitation in cell‐free by mapping the chemical space of ten low‐cost additives with four distinct mechanisms of action in a combinatorial manner to elucidate their capacity to further stabilize lyophilized cell extract. We report three key findings: 1) unique additive formulations that maintain full productivity of the cell extract at 4°C and 23°C; 2) additive formulations that enhance extract productivity up to 195%; 3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations that were not tested experimentally. The identified additive formulations provide a simple and low‐cost advance toward making CFPS field‐ready.Support or Funding InformationBill and Linda Frost Fund, Center for Applications in Biotechnology’s Chevron Biotechnology Applied Research Endowment Grant, Cal Poly Research, Scholarly, and Creative Activities Grant Program (RSCA 2017), and the National Science Foundation (NSF‐1708919)Comparison of traditional cellfree extract production and reaction setup with our modified workflow for improved stability and productivity to enable field applications.Figure 1

Chaperone proteins as single component reagents to assess antibody nonspecificity

ABSTRACTEarly stage assays that evaluate monoclonal antibody drug-like properties serve as valuable tools for selection of lead candidates. One liability for clinical development, off-target reactivity, is often assessed by binding to a mixture or panel of noncognate proteins. While robust, these mixes are often ill-defined, and can suffer from issues such as lot-to-lot variability. In this study, we discovered in immunoprecipitation experiments that certain chaperones are present in one of these mixtures;we then explored the use of recombinant chaperone proteins as well-characterized agents to predict antibody nonspecificity. Antibody binding to the heat shock proteins HSP70, HSP90, or trigger factor all served as predictors of cross-interaction propensity, with HSP90 providing the greatest ability to predict antibody clearance rates in mouse. Individual chaperone binding correlates surprisingly closely with binding to complex cell extracts, with the exception of a few “false negatives” (assuming a complex cell extract as the “true” value). As defined reagents, these chaperone reagents present advantages for high throughput assays of nonspecificity.

Nucleotide and Nucleotide Sugar Analysis in Cell Extracts by Capillary Electrophoresis.

In biotechnological processes the intracellular level of nucleotides and nucleotide sugars have a direct impact on the post-translational modification (glycosylation) of the therapeutic protein products and on the exopolysaccharide pattern of the cells. Thus, they are precursors and also key components in the production of glycoproteins and glycolipids. All four nucleotides (at different phosphorylation stages) and their natural sugar derivatives coexist in biological samples. Their relative ratios depend on the actual conditions under which the cells are grown. Therefore, their simultaneous determination at different time points and different cell culture conditions in biotechnological samples is of interest in order to develop the optimal cell culture process. In our study capillary electrophoresis (CE) combined with UV detection @ 260 nm was selected for the separation and quantification of the complex nucleotide mixture of the structurally very similar nucleotides and nucleotide sugars in cell extracts. The high separation efficiency of CE as well as its insensitivity to the complex cell matrix makes this method superior to commonly used HPLC methods. In our study eleven nucleotides and six nucleotide sugars were analyzed. A robust and reproducible analysis system was developed. As background electrolyte borate (40 mM, pH 9.5) was used containing 1% PEG (MW 35'000 Da) which enhanced resolution. In order to obtain high reproducibility in terms of migration time, mandatory for the unambiguous identification of the single compounds in the complex cell extract mixtures, dynamic coating was also employed. The method was tested for CHO cell extracts where three sugar nucleotides and seven nucleotides were identified and quantified using GDP-Glc as internal standard.

Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling.

Small ubiquitin-related modifiers (SUMO) are evolutionarily conserved ubiquitin-like proteins that regulate several cellular processes including cell cycle progression, intracellular trafficking, protein degradation and apoptosis. Despite the importance of protein SUMOylation in different biological pathways, the global identification of acceptor sites in complex cell extracts remains a challenge. Here we generate a monoclonal antibody that enriches for peptides containing SUMO remnant chains following tryptic digestion. We identify 954 SUMO3-modified lysine residues on 538 proteins and profile by quantitative proteomics the dynamic changes of protein SUMOylation following proteasome inhibition. More than 86% of these SUMOylation sites have not been reported previously, including 5 sites on the tumour suppressor parafibromin (CDC73). The modification of CDC73 at K136 affects its nuclear retention within PML nuclear bodies on proteasome inhibition. In contrast, a CDC73 K136R mutant translocates to the cytoplasm under the same conditions, further demonstrating the effectiveness of our method to characterize the dynamics of lysine SUMOylation.

Specific visualization and identification of phosphoproteome in gels.

The applicability of gel-based proteomic strategies in phosphoproteomics has been largely limited by the lack of technologies for specific detection of phosphoproteins in gels. Here for the first time we report a strategy for simultaneous visualization and identification of phosphoproteome in gels (VIPing) through coupling specific detection of phosphoproteins with protein identification and phosphorylation site mapping by tandem mass spectrometry. The core of the strategy is a novel compound multifunctionalized with a titanium ion(IV) for outstanding selectivity toward phosphorylated residues, a fluorophore for visualization, and a biotin group for phosphopeptide enrichment. The sensitivity and specificity of the VIPing strategy was demonstrated using standard protein mixtures and complex cell extracts, and the method was applied to study the phosphorylation changes of an essential tyrosine kinase Syk and interacting proteins upon B-cell stimulation. The novel technique provides a powerful platform for gel-based phosphoproteomic studies.

Targeted Identification of SUMOylation Sites in Human Proteins Using Affinity Enrichment and Paralog-specific Reporter Ions

Protein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification of SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping SUMOylated Lys residues by mass spectrometry (MS) is challenging because of the dynamic nature of this modification, the existence of three functionally distinct human SUMO paralogs, and the large SUMO chain remnant that remains attached to tryptic peptides. To overcome these problems, we created HEK293 cell lines that stably express functional SUMO paralogs with an N-terminal His6-tag and an Arg residue near the C terminus that leave a short five amino acid SUMO remnant upon tryptic digestion. We determined the fragmentation patterns of our short SUMO remnant peptides by collisional activation and electron transfer dissociation using synthetic peptide libraries. Activation using higher energy collisional dissociation on the LTQ-Orbitrap Elite identified SUMO paralog-specific fragment ions and neutral losses of the SUMO remnant with high mass accuracy (< 5 ppm). We exploited these features to detect SUMO modified tryptic peptides in complex cell extracts by correlating mass measurements of precursor and fragment ions using a data independent acquisition method. We also generated bioinformatics tools to retrieve MS/MS spectra containing characteristic fragment ions to the identification of SUMOylated peptide by conventional Mascot database searches. In HEK293 cell extracts, this MS approach uncovered low abundance SUMOylated peptides and 37 SUMO3-modified Lys residues in target proteins, most of which were previously unknown. Interestingly, we identified mixed SUMO-ubiquitin chains with ubiquitylated SUMO proteins (K20 and K32) and SUMOylated ubiquitin (K63), suggesting a complex crosstalk between these two modifications.

Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.

BackgroundHigh throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities.Methodology and Principal FindingsHere, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z’>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates.ConclusionsWe believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

Artifactual Sulfation of Silver-stained Proteins

Sulfation and phosphorylation are post-translational modifications imparting an isobaric 80-Da addition on the side chain of serine, threonine, or tyrosine residues. These two post-translational modifications are often difficult to distinguish because of their similar MS fragmentation patterns. Targeted MS identification of these modifications in specific proteins commonly relies on their prior separation using gel electrophoresis and silver staining. In the present investigation, we report a potential pitfall in the interpretation of these modifications from silver-stained gels due to artifactual sulfation of serine, threonine, and tyrosine residues by sodium thiosulfate, a commonly used reagent that catalyzes the formation of metallic silver deposits onto proteins. Detailed MS analyses of gel-separated protein standards and Escherichia coli cell extracts indicated that several serine, threonine, and tyrosine residues were sulfated using silver staining protocols but not following Coomassie Blue staining. Sodium thiosulfate was identified as the reagent leading to this unexpected side reaction, and the degree of sulfation was correlated with increasing concentrations of thiosulfate up to 0.02%, which is typically used for silver staining. The significance of this artifact is discussed in the broader context of sulfation and phosphorylation site identification from in vivo and in vitro experiments.

Evaluation of commercial EBV RecombLine assay for diagnosis of nasopharyngeal carcinoma

Background In recent years a number of Epstein–Barr virus (EBV) proteins were defined as being immunodominant for either IgM, IgG or IgA immune responses, yielding promising markers for diagnostic serology. Specific reactivity patterns to these proteins have been described for infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC), various types of lymphoma, and healthy EBV carriers. Objectives To compare the NPC-related diagnostic value of EBV RecombLine test (Mikrogen, Germany) with a standardized immunoblot assay [Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM, et al. Molecular diversity of Epstein–Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004;190:53–62] and to define the diagnostic value of individual EBV marker proteins in a population with high incidence of NPC. Result Sera from Indonesian NPC patients taken at primary diagnosis ( n = 108) were analyzed for IgG and IgA reactivity and compared with regional healthy blood donors ( n = 62), non-NPC patient controls ( n = 10) and IM patients ( n = 10). Most NPC patients and controls showed strong IgG reactivity to VCA-p18, -p23, and EBNA1, limiting their diagnostic use. Few (<20%) healthy donors and patient controls showed IgG reactivity to EA proteins p47/54 and p138, yielding combined sensitivity/specificity and PPV/NPV values of 92.6%/98.3% and 99.0%/88.1%, for diagnosing NPC. NPC sera showed significantly more EBV reactive IgA antibody (>80% positive) than controls (<10% positive), although being less broadly reactive and significantly less strong compared to IgG. For IgA best results were observed for RecombLine EBNA1 with sensitivity/specificity and PPV/NPV values of 92%/89% and 93.4%/85.9%, respectively. Conclusion In high incidence NPC regions with low incidence IM yet high prevalence of EBV infection, both RecombLine IgG and IgA tests provide a useful alternative to the more complex cell-extract based immunoblot assay as confirmation test for NPC diagnosis in particular when using EA and EBNA1 as discriminators in IgG and IgA testing, respectively.

Optimizing Thiophosphorylation in the Presence of Competing Phosphorylation with MALDI-TOF−MS Detection

Thiophosphorylation provides a metabolically stable, chemically reactive phosphorylation analogue for analyzing the phosphoproteome in vitro and in vivo. We developed a MALDI-TOF-MS based assay for optimizing thiophosphopeptide production by a kinase even in the presence of Mg(2+) and ATP. We found that Abl kinase thiophosphorylation rates can be "rescued" using Mn(2+) in the presence of Mg(2+). Under our ideal conditions, titration of Mn(2+) and ATPgammaS in the presence of Mg(2+) allowed relatively rapid, highly specific thiophosphorylation by Abl tyrosine kinase, both as purified enzyme and in complex cell extracts.

Proteomic Analysis of Calcium/Calmodulin-dependent Protein Kinase I and IV in Vitro Substrates Reveals Distinct Catalytic Preferences

The multifunctional calcium/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV) are closely related by primary sequence and predicted to have similar substrate specificities based on peptide studies. We identified a fragment of p300-(1-117) that is a substrate of both kinases, and through both mutagenesis and Edman phosphate ((32)P) release sequencing, established that CaMKI and CaMKIV phosphorylate completely different sites. The CaMKI site, Ser(89) ((84)LLRSGSSPNL(93)), fits the expected consensus whereas the CaMKIV site, Ser(24) ((19)SSPALSASAS(28)), is novel. To compare kinase substrate preferences more generally, we employed a proteomic display technique that allowed comparison of complex cell extracts phosphorylated by each kinase in a rapid in vitro assay, thereby demonstrating substrate preferences that overlapped but were clearly distinct. To validate this approach, one of the proteins labeled in this assay was identified by microsequencing as HSP25, purified as a recombinant protein, and examined as a substrate for both CaMKI and CaMKIV. Again, CaMKI and CaMKIV were different, this time in kinetics and stoichiometry of the phosphorylation sites, with CaMKI preferring Ser(15) ((10)LLRTPSWGPF(19)) to Ser(85) ((80)LNRQLSSGVS(89)) 3:1, but CaMKIV phosphorylating the two sites equally. These differences in substrate specificities emphasize the need to consider these protein kinases independently despite their close homology.

Specific Complex of Human Immunodeficiency Virus Type 1 Rev and Nucleolar B23 Proteins: Dissociation by the Rev Response Element

The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.

Specific complex of human immunodeficiency virus type 1 rev and nucleolar B23 proteins: dissociation by the Rev response element.

The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.

Measurement of δ-( l-α-aminoadipyl)- l-cysteinyl- d-valine synthetase activity in Streptomyces clavuligerus by high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde

A procedure for rapid, sensitive measurement of δ-( l-α-aminoadipyl)- l-cysteinyl- d-valine (ACV) in complex cell extracts has been developed by adapting a widely used method for high-performance liquid chromatography analysis of amino acids. Samples were oxidized with performic acid and derivatized with o-phthaldialdehyde-mercaptoethanol to give the fluorescent isoindole derivative of the peptide sulfonate. The procedure was used to assay Strptomyces clavuligerus cell extracts and partially purified fractions for ACV synthetase activity and to determine some characteristics of the enzyme reaction. The presence of a second enzyme with cysteine as a substrate was also indicated.