Determination of the biological origin of enzyme preparations using SDS-PAGE and peptide mass fingerprinting.

Enzymes are mainly extracted from the culture broth of microorganisms. Various commercially available enzyme preparations (EPs) are derived from different microorganisms, and the source of the EP should be the same as that mentioned in the manufacture's information. The development of analytical methods that can determine the origin of the final products is important for ensuring that the EPs are nontoxic, especially when used as food additives. In this study, various EPs were subjected to SDS-PAGE, and the main protein bands were excised. After in-gel digestion, the generated peptides were analysed using MALDI-TOF MS, and protein identification was performed by searching the set of peptide masses against protein databases. In total, 36 EPs including amylase, ß-galactosidase, cellulase, hemicellulase and protease were analysed, and the information about the enzyme sources was obtained for 30 EPs. Among these, the biological sources determined for 25 EPs were consistent with the manufacturer's information; for the remaining five, enzymes produced by closely-related species were shown as matching proteins due to high sequence similarity. Six enzymes derived from four microorganisms could not be identified because their protein sequences were not registered in the database. As these databases are expanded, this approach of using SDS-PAGE and peptide mass fingerprinting (PMF) can determine the biological origin of enzymes rapidly and contribute to ensuring the safety of EPs.

Combinatorial Enzymatic Catalysis for Bioproduction of Ginsenoside Compound K.

Ginsenoside compound K (CK) is an emerging functional food or pharmaceutical product. To date, there are still challenges to exploring effective catalytic enzymes for enzyme-catalyzed manufacturing processes and establishing enzyme-catalyzed processes. Herein, we identified three ginsenoside hydrolases BG07 (glucoamylase), BG19 (ß-glucosidase), and BG23 (ß-glucosidase) from Aspergillus tubingensis JE0609 by transcriptome analysis and peptide mass fingerprinting. Among them, BG23 was expressed in Komagataella phaffii with a high volumetric activity of 235.73 U mL-1 (pNPG). Enzymatic property studies have shown that BG23 is an acidic (pH adaptation range of 4.5-7.0) and mesophilic (thermostable < 50 °C) enzyme. Moreover, a one-pot combinatorial enzyme-catalyzed strategy based on BG23 and BGA35 (ß-galactosidase from Aspergillus oryzae) was established, with a high CK yield of 396.7 mg L-1 h-1. This study explored the ginsenoside hydrolases derived from A. tubingensis at the molecular level and provided a reference for the efficient production of CK.

[Proteome Analysis for Identification of the Origin of Biological Foreign Substances].

We have developed a method for determination of the species of origin of biological foreign substances using proteomics analysis technology. That is, the amino acid sequence of the tryptic digested product of the protein extracted from the foreign substance is determined by high-resolution LC-MS, and the amino acid sequence is collated with a public protein database to determine the origin species of the foreign substance. As a result of testing meat (beef, pork, chicken) and egg (chicken, quail) from known origin as simulated foreign substances, we were able to find species-specific amino acid sequences for each species, suggesting that the developed method is useful for species discrimination of a foreign substances that have been heat-treated with retort, this method is potentially useful to complement DNA analysis, for determination of the species of origin of foreign substances.

Laboratory Diagnostic Methods and Antibiotic Resistance Patterns of Staphylococcus aureus and Escherichia coli Strains: An Evolving Human Health Challenge.

Raw ground meat is known as a transmission vehicle for biological agents that may be harmful to human health. The objective of the present study was to assess microbiological quality of the ground meats. A total of 280 samples of local and imported chilled meats were randomly collected from retail shops in Buraydah City, Saudi Arabia. The meat samples were microbiologically analyzed using standard methods, peptide mass fingerprinting (PMF) technique, MicroScan Walkaway System (MicroScan) and qPCR System. The imported meat was more bacterially contaminated than local meat, with variable contamination degrees of Staphylococcus aureus (40.33%), Escherichia coli (36.13%), Hafnia alvei (7.56%), Pseudomonas spp. (6.72%), Salmonella spp. (5.88%) and Aeromonas spp. (3.36%). PMF verified all the isolated bacteria by 100%, compared to 75-95% achieved by MicroScan. The gene encoding flagellin (fliC) was recognized in 67.44% of E. coli strains, while the thermonuclease (nuc) and methicillin resistance (mecA) genes were detected in 100% S. aureus and 39.6% of methicillin-resistant S. aureus (MRSA) strains, respectively. The S. aureus and E. coli strains were highly resistant to multiple antibiotics (e.g., ampicillin, amoxicillin-clavulanic acid and cephalothin). For identifying various foodborne pathogens, PMF has been recognized as a powerful and precise analytical method. In light of the increasing use of PMF to detect multidrug-resistant bacteria, this study emphasizes the need for improved ways of treating and preventing pathogens, as well as setting up monitoring systems to guarantee hygiene and safety in meat production.

Bottom-Up Analysis of Proteins by Peptide Mass Fingerprinting with tCITP-CZE-ESI-TOF MS After Tryptic Digest.

The identification of proteins in samples of moderate to complex composition is primarily done by bottom-up approaches. Therefore, proteins are enzymatically digested, mostly by trypsin, and the resulting peptides are then separated prior to their transfer to a mass spectrometer. The following protocol portrays a bottom-up method, which was optimized for the application of CZE-ESI-TOF MS. Protein denaturation is achieved by addition of 2,2,2-trifluoroethanol (TFE) and heat treatment. Afterwards, disulfide bonds are reduced with tris-(2-carboxyethyl)phosphine (TCEP) and subsequently alkylated with iodoacetamide (IAA). The tryptic digest is performed in an ammonium bicarbonate buffer at pH 8.0. The digested protein sample is then concentrated in-capillary by transient capillary isotachophoresis (tCITP) with subsequent CZE separation of tryptic peptides in an acidic background electrolyte. Hyphenation to a time-of-flight (TOF) mass spectrometer is carried out by a triple-tube coaxial sheath flow interface, which uses electrospray ionization (ESI). Peptide identification is done by peptide mass fingerprinting (PMF). The protocol is outlined exemplarily for a model protein, i.e., bovine ß-lactoglobulin A.

A primer for ZooMS applications in archaeology.

Collagen peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, also known as zooarchaeology by mass spectrometry (ZooMS), is a rapidly growing analytical technique in the fields of archaeology, ecology, and cultural heritage. Minimally destructive and cost effective, ZooMS enables rapid taxonomic identification of large bone assemblages, cultural heritage objects, and other organic materials of animal origin. As its importance grows as both a research and a conservation tool, it is critical to ensure that its expanding body of users understands its fundamental principles, strengths, and limitations. Here, we outline the basic functionality of ZooMS and provide guidance on interpreting collagen spectra from archaeological bones. We further examine the growing potential of applying ZooMS to nonmammalian assemblages, discuss available options for minimally and nondestructive analyses, and explore the potential for peptide mass fingerprinting to be expanded to noncollagenous proteins. We describe the current limitations of the method regarding accessibility, and we propose solutions for the future. Finally, we review the explosive growth of ZooMS over the past decade and highlight the remarkably diverse applications for which the technique is suited.

MALDI-TOF-MS-Based Identification of Monoclonal Murine Anti-SARS-CoV-2 Antibodies within One Hour.

During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.

Sequence determination and bioinformatic comparison of ten venom serine proteases of Trimeresurus gracilis, a Taiwanese endemic pitviper with controversial taxonomy.

Trimeresurus gracilis (Tgc) is endemic to Taiwan and shown to be closely related with Ovophis okinavensis by previous phylogenetic analyses, but their taxonomic status remain controversial. Here, we cloned and sequenced ten of its venom serine-proteases (designated as Tgc-vSPs). All the Tgc-vSPs conserve the catalytic triads, six appear to be kallikrein-like (KNs) and four are plasminogen-activator homologs (PAHs and PAs). They are studied under four structural categories: (1) highly similar Tgc-KN1, Tgc-KN2 and Tgc-KN3, with four predicted N-glycosylation sites; (2) Tgc-KN4, with a single N -glycosylation site; (3) Tgc-KN5 and Tgc-KN6, with two distinct N-glycosylation sites; (4) Tgc-PAH1/PAH2, TgcPA3, and Tgc-PA4, with two conserved N-glycosylation sites. Additionally, Tgc-KN1, Tgc-KN4 and Tgc-PAH1 were purified by reversed-phase HPLC and identified by peptide-mass-fingerprinting. Results of BLAST and sequence alignments reveal that Tgc-KN1∼3 and Tgc-KN6 are most like the vSPs of rattlesnakes, while the sequences of Tgc-KN4, KN5 and Tgc-PAH1/PAH2 match closely to the partial sequences of three O. okinavensis vSPs. Thus, our results reveal non-overlapping similarities of Tgc-vSPs to the O. okinavensis vSPs and vSPs of the New World pitvipers. In addition, molecular phylogenetic analyses of the plasminogen-activator like vSPs reveal separate evolution of two clusters of the enzymes with distinct functions.

Atrazine detoxification by intracellular crude enzyme extracts derived from epiphytic root bacteria associated with emergent hydrophytes.

The present study demonstrated atrazine detoxification by intracellular crude enzyme extracts of Pseudomonas spp. strains ACB and TLB. Indigenous bacterial protein-based remediation techniques could be an alternative to bioaugmentation which pose multiple challenges when applied to the field. Intracellular enzymes were extracted from strains ACB and TLB and their degradation potential of 10 mg L-1 was determined using Gas Chromatography; further, enzyme extracts were subjected to protein profiling studies. In span of 6 h, enzyme extracts of strain ACB showed maximum degradation at 30 °C and 40 °C (71%) and enzyme extracts of strain TLB showed maximum degradation at 40 °C (48%). Atrazine degradation by enzyme extracts of strain ACB showed maximum degradation at pH 7 (71%) and pH 6 (69%) in 6 h. Similarly, enzyme extracts of strain TLB showed maximal degradation at pH 6 (46%) in 6 h. The present study demonstrated, for the first time, efficient atrazine remediation by intracellular crude enzyme extracts from epiphytic root bacteria at a range of temperature and pH conditions. Protein profiling studies indicated that atrazine induced expression of CoA ester lyase and alkyl hydroperoxide reductase in the strains ACB and TLB respectively. Expressions of these proteins have never been associated with atrazine exposure.

Molecular mapping of platelet hyperreactivity in diabetes: the stress proteins complex HSPA8/Hsp90/CSK2α and platelet aggregation in diabetic and normal platelets.

The molecular understanding of the pathophysiological changes elicited by diabetes in platelets may help in further elucidating the involvement of this pseudo-cell in the increased risk of developing cardiovascular disease and thrombosis in diabetic subjects. We aimed to investigate the differential characteristics of platelets from diabetic patients and nondiabetic controls to unveil the molecular mechanisms behind the increased platelet reactivity in diabetes. We compared platelets from diabetic and control subjects by 2 dimensional-electrophoresis followed by mass spectrometry. Changes in selected differential proteins were validated by immunoprecipitation assays and western blot. Platelet aggregation was measured by light transmittance aggregometry induced by collagen and ADP, and dynamic coagulation analysis of whole blood was measured by thromboelastometry. We observed significant differences in proteins related to platelet aggregation, cell migration, and cell homeostasis. Subjects with diabetes showed higher platelet aggregation and thrombogenicity and higher contents of the stress-related protein complex HSPA8/Hsp90/CSK2α than nondiabetic subjects. Changes in the chaperones HSPA8 and Hsp90, and in CSK2α protein contents correlated with changes in platelet aggregation and blood coagulation activity. In conclusion, the complex HSPA8/Hsp90/CSK2α is involved in diabetes-related platelet hyperreactivity. The role of the HSPA8/Hsp90/CSK2α complex may become a molecular target for the development of future preventive and therapeutic strategies for platelet dysfunction associated with diabetes and its complications.

Serum IgM heavy chain sub-isotypes and light chain variants revealed in giant grouper (Epinephelus lanceolatus) via protein A affinity purification, mass spectrometry and genome sequencing.

Two IgM heavy (H) chain sub-isotypes (80 and 40 kDa) and two light (L) chain variants (25 and 30 kDa) were detected in the serum of giant grouper (Epinephelus lanceolatus), purified by ammonium sulphate precipitation followed by protein A affinity chromatography. This method yielded 5.6 mg/mL high purity IgM from grouper serum, with efficiency estimated at 39.5% recovery from crude serum. The H and L chains were identified by SDS-PAGE and mass spectrometry (MS). Nanopore long-read sequencing was used to generate a genomic contig (MW768935), containing Cµ, Cδ loci, VH regions, and a H chain Joining segment. cDNA sequencing of Cµ transcripts (MW768933 and MW768934) were used to polish the genomic contig and determine the exons and introns of the corresponding locus. MS peptide mapping revealed that the 80 kDa H chain consisted of CH1-4 domains while peptides from the 40 kDa H chain only mapped to CH1-2 domains. Our genomic contig showed the Cµ locus has a Cµ1-Cµ2-Cµ3-Cµ4 arrangement on the same strand as the other Ig loci identified in this genomic sequence. Our study corrects the NCBI annotations of the opposing Cµ loci (LOC117268697 and LOC117268550) in chromosome 16 (NC_047006). Further, we identified both κ and λ L chain isotypes in serum IgM. The molecular weight differences observed may result from different combinations of CL and VL genes. Putative IgM sub-isotypes have also been reported in Epinephelus itajara and Epinephelus coioides. The presence of IgM sub-isotypes may be a conserved trait among Epinephelus species.

nCOV-19 peptides mass fingerprinting identification, binding, and blocking of inhibitors flavonoids and anthraquinone of Moringa oleifera and hydroxychloroquine.

An rare pandemic of viral pneumonia occurs in December 2019 in Wuhan, China, which is now recognized internationally as Corona Virus Disease 2019 (COVID-19), the etiological agent classified as Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). According to the World Health Organization (WHO), it has so far expanded to more than 213 countries/territories worldwide. Our study aims to find the viral peptides of SARS-COV-2 by peptide mass fingerprinting (PMF) in order to predict its novel structure and find an inhibitor for each viral peptide. For this reason, we calculated the mass of amino acid sequences translated from the SARS-CoV2 whole genome and identify the peptides that may be a target for inhibition. Molecular peptide docking with Moringa oleifera, phytochemicals (aqueous and ethanolic) leaf extracts of flavonoids (3.56 ± 0.03), (3.83 ± 0.02), anthraquinone (11.68 ± 0.04), (10.86 ± 0.06) and hydroxychloroquine present therapy of COVID-19 in Pakistan for comparative study. Results indicate that 15 peptides of SARS-CoV2 have been identified from PMF, which is then used as a selective inhibitor. The maximum energy obtained from AutoDock Vina for hydroxychloroquine is -5.1 kcal/mol, kaempferol (flavonoid) is -6.2 kcal/mol, and for anthraquinone -6 kcal/mol. Visualization of docking complex, important effects are observed regarding the binding of peptides to drug compounds. In conclusion, it is proposed that these compounds are effective antiviral agents against COVID-19 and can be used in clinical trials.Communicated by Ramaswamy H. Sarma.

Proteomic Analysis of Medicinal Plant Calotropis Gigantea by In Silico Peptide Mass Fingerprinting.

Medicinal plants are the basic source of medicinal compounds traditionally used for the treatment of human diseases. Calotropis gigantea is a medicinal plant belonging to the family of Apocynaceae in the plant kingdom and subfamily Asclepiadaceae usually bearing multiple medicinal properties to cure a variety of diseases. BACKGROUND: The Peptide Mass Fingerprinting (PMF) identifies the proteins from a reference protein database by comparing the amino acid sequence that is previously stored in the database and identified. OBJECTIVE: The purpose of the study is to identify the peptides having anti-cancerous properties by in silico peptide mass fingerprinting. METHODS: The calculation of in silico peptide masses is done through the ExPASy PeptideMass and these masses are used to identify the peptides from the MASCOT online server. Anticancer probability is calculated by iACP server, docking of active peptides is done by CABS-dock the server. RESULTS: The anti-cancer peptides are identified with the MASCOT peptide mass fingerprinting server, the identified peptides are screened and only the anti-cancer are selected. De-novo peptide structure prediction is used for 3D structure prediction by PEP-FOLD 3 server. The docking results confirm strong bonding with the interacting amino acids of the receptor protein of breast cancer BRCA1 which shows the best peptide binding to the active chain, the human leukemia protein docking with peptides shows the accurate binding. CONCLUSION: These peptides are stable and functional and are the best way for the treatment of cancer and many other deadly diseases.

A Novel Reversibly Glycosylated Polypeptide-2 of Bee Pollen from Rape (Brassica napus L.): Purification and Characterization.

BACKGROUND: Reversibly glycosylated polypeptide (RGP), a kind of hydrosoluble and plasmodesmal-associated protein found in plants, plays a crucial role in the development of pollen. OBJECTIVE: A novel RGP 2 was isolated and identified from rape (Brassica napus L.) bee pollen. METHODS: RGP2 was isolated and purified by ion-exchange column and gel filtration chromatography, and characterized by MALDI-TOF-MS, LC-MS, immunological histological chemistry, and transmission electron microscope. RESULTS: Our results indicated that the RGP2 is an acidic protein (pI=5.46) with the molecular weight 42388 Da. It contained 17 kinds of amino acids, among which aspartic acid had the highest amount (71.56 mg/g). Homologous alignment of amino acid sequence results showed that RGP2 was 80.33%, 85.02%, 86.06%, and 88.93% identical to Arabidopsis thaliana RGP2 (AtRGP2), Oryza sativa RGP (OsRGP), Triticum aestivum RGP (TaRGP), and Zea maize RGP (ZmRGP), respectively. The localization results showed that RGP2 in rape anther existed in exine and intine of anther cells of rape flower by immunological histological chemistry and the subcellular localization identified that RGP2 appeared around the Golgi apparatus in cytoplasm by transmission electron microscope. CONCLUSION: RGP2 has a highly conserved sequence of amino acid residues and potential glycosylation sites.

On the standardization of ZooMS nomenclature.

Zooarchaeology by Mass Spectrometry (ZooMS) is rapidly becoming a staple in archaeological and cultural heritage science. Developed a decade ago, this peptide mass fingerprinting technique is expanding from a small group of researchers mainly involved in method development to a broader group of scientists using it as another tool in their toolboxes. With new researchers beginning to use the method, it is imperative that a user-friendly, standardized approach be established. A major barrier has been the often haphazard and changing nomenclature used to label peptide markers necessary for taxonomic identification. Consistent, reliable, and easy-to-understand nomenclature is key to the growth of ZooMS, particularly as the reference library continues to expand. We propose a new set of standardized guidelines for peptide markers based on their position in the type I collagen sequence from the beginning of the highly conserved, helical region. Since this region has no insertions or deletions over a wide range of taxonomic groups, the proposed nomenclature system can be used reliably and consistently across all vertebrate taxa. We propose to label ZooMS peptide markers with the gene, followed by the position of the first and last amino acid of the marker from the start of the helical region. SIGNIFICANCE STATEMENT: We propose a standardized nomenclature system for ZooMS peptide markers that provides consistent labels across multiple, broad taxonomic groups. This system unambiguously locates the marker peptides in the type I collagen sequence, avoids duplication of marker names, and facilitates the creation of large ZooMS databases which can include all vertebrates.

Update on Proteomic approaches to uncovering virus-induced protein alterations and virus -host protein interactions during the progression of viral infection.

INTRODUCTION: Viruses induce profound changes in the cells they infect. Understanding these perturbations will assist in designing better therapeutics to combat viral infection. System-based proteomic assays now provide unprecedented opportunity to monitor large numbers of cellular proteins. AREAS COVERED: This review will describe various quantitative and functional mass spectrometry-based methods, and complementary non-mass spectrometry-based methods, such as aptamer profiling and proximity extension assays, and examples of how each are used to delineate how viruses affect host cells, identify which viral proteins interact with which cellular proteins, and how these change during the course of a viral infection. PubMed was searched multiple times prior to manuscript submissions and revisions, using virus, viral, proteomics; in combination with each keyword. The most recent examples of published works from each search were then analyzed. EXPERT OPINION: There has been exponential growth in numbers and types of proteomic analyses in recent years. Continued development of reagents that allow increased multiplexing and deeper proteomic probing of the cell, at quantitative and functional levels, enhancements that target more important protein modifications, and improved bioinformatics software tools and pathway prediction algorithms will accelerate this growth and usher in a new era of host proteome understanding.

Cellular response of Brevibacterium casei #NIOSBA88 to arsenic and chromium-a proteomic approach.

Cellular response against different heavy metal stress differs with the metal. Arsenic and chromium are heavy metals and toxic to living systems. The concentration of these metals in seawater is very low. However, due to their solubility in nature, they actively enter cells via various transport mechanisms and cause damage to the cells. Brevibacterium casei #NIOSBA88, a marine-derived, gram-positive isolate was multi-metal tolerant. Proteomic analysis of this isolate in response to arsenic and chromium resulted in the identification of total 2549 proteins, out of which 880 proteins were found to be commonly expressed at 750 mgL-1 arsenic and 100 mgL-1 chromium and in absence of both the metals. In contrast, 533, 212, and 270 proteins were found to be unique in the absence of any metal, 750 mgL-1 of arsenic and 100 mgL-1 of chromium respectively. Proteins such as antibiotic biosynthesis monooxygenase, ArsR family transcriptional regulator, cytochrome C oxidase subunit II, and thioredoxin reductase were exclusively expressed only in response to arsenic and chromium. Other proteins like superoxide dismutase, lipid hydroperoxide reductase, and thioredoxin-disulfide reductase were found to be upregulated in response to both the metals. Most of the proteins involved in the normal cell functioning were found to be downregulated. Major metabolic functions affected include amino acid metabolism, carbohydrate metabolism, translation, and energy metabolism. Peptide mass fingerprinting of Brevibacterium casei #NIOSBA88 exposed to arsenic and chromium respectively revealed the deleterious effect of these metals on the bacterium and its strategy to overcome the stress.

Dataset on phenotypic characterization, on protein and genome analysis of three fluorescent Pseudomonas strains from mid-mountain water.

The identification of non-fermentative Gram negative bacilli from run-off and spring water, including fluorescent Pseudomonas is very complex and investigations are needed to contribute to the systematic of these bacteria. In this dataset, the phenotypical profiles of three strains isolated from Vosges mountains first identified as Pseudomonas fluorescens were determined using APIⓇ 50 CH galleries. Then, the identification of their proteins released directly into water was carried out using tandem/mass spectrometry after separating proteins on native two-dimensional polyacrylamide gels. Finally, genotypic analysis data is presented, that illustrates biodiversity in this fluorescent bacterial group. This data is referred by a research article entitled "Fluorescent Pseudomonas strains from mid-mountain water able to release antioxidant proteins directly into water".

Quantitative Proteomics of Uukuniemi Virus-host Cell Interactions Reveals GBF1 as Proviral Host Factor for Phleboviruses.

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.

A novel approach for protein identification from complex cell proteome using modified peptide mass fingerprinting algorithm.

A unique peptide based search algorithm for identification of protein mixture using PMF is proposed. The proposed search algorithm utilizes binary search and heapsort programs to generate frequency chart depicting the unique peptides corresponding to all proteins in a proteome. The use of binary search program significantly reduces the time for frequency chart preparation to ∼2 s for a proteome comprising ∼23 000 proteins. The algorithm was applied to a three-protein mixture identification, host cell protein (HCP) analysis, and a simulation-generated data set. It was found that the algorithm could identify at least one unique peptide of a protein even in the presence of fourfold higher concentration of another protein. In addition, two HCPs that are known to be difficult to remove were missed by MS/MS approach and were exclusively identified using the presented algorithm. Thus, the proposed algorithm when used along with standard proteomic approaches present avenues for enhanced protein identification efficiency, particularly for applications such as HCP analysis in biopharmaceutical research, where identification of low-abundance proteins are generally not achieved due to dynamic range limitations between the target product and HCPs.