Label-free Quantitative Mass Spectrometry Research Articles

Proteomics Insights into the Biomass Hydrolysis Potentials of a Hypercellulolytic Fungus Penicillium funiculosum.

The quest for cheaper and better enzymes needed for the efficient hydrolysis of lignocellulosic biomass has placed filamentous fungi in the limelight for bioprospecting research. In our search for efficient biomass degraders, we identified a strain of Penicillium funiculosum whose secretome demonstrates high saccharification capabilities. Our probe into the secretome of the fungus through qualitative and label-free quantitative mass spectrometry based proteomics studies revealed a high abundance of inducible CAZymes and several nonhydrolytic accessory proteins. The preferential association of these proteins and the attending differential biomass hydrolysis gives an insight into their interactions and clues about possible roles of novel hydrolytic and nonhydrolytic proteins in the synergistic deconstruction of lignocellulosic biomass. Our study thus provides the first comprehensive insight into the repertoire of proteins present in a high-performing secretome of a hypercellulolytic Penicillium funiculosum, their relative abundance in the secretome, and the interaction dynamics of the various protein groups in the secretome. The gleanings from the stoichiometry of these interactions hold a prospect as templates in the design of cost-effective synthetic cocktails for the optimal hydrolysis of biomass.

Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry

Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography–tandem mass spectrometry (LC–MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMSE) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC–MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest.

Proteomic Profiling of Hematopoietic Stem/Progenitor Cells after a Whole Body Exposure of CBA/CaJ Mice to Titanium (48Ti) Ions.

Myeloid leukemia (ML) is one of the major health concerns from exposure to radiation. However, the risk assessment for developing ML after exposure to space radiation remains uncertain. To reduce the uncertainty in risk prediction for ML, a much increased understanding of space radiation-induced changes in the target cells, i.e., hematopoietic stem/progenitor cells (HSPCs), is critically important. We used the label-free quantitative mass spectrometry (LFQMS) proteomic approach to determine the expression of protein in HSPC-derived myeloid colonies obtained at an early time-point (one week) and a late time-point (six months) after an acute whole body exposure of CBA/CaJ mice to a total dose of 0, 0.1, 0.25, or 0.5 Gy of heavy-ion titanium (48Ti ions), which are the important component of radiation found in the space environment. Mice exposed to 0 Gy of 48Ti ions served as non-irradiated sham controls. There were five mice per treatment groups at each harvest time. The Trans-Proteomic Pipeline (TPP) was used to assign a probability of a particular protein being in the sample. A proof-of-concept based Ingenuity Pathway Analysis (IPA) was used to characterize the functions, pathways, and networks of the identified proteins. Alterations of expression levels of proteins detected in samples collected at one week (wk) post-irradiation reflects acute effects of exposure to 48Ti ions, while those detected in samples collected at six months (mos) post-irradiation represent protein expression profiles involved in the induction of late-occurring damage (normally referred to as genomic instability). Our results obtained by using the IPA analyses indicate a wide array of signaling pathways involved in response to 1 GeV/n 48Ti ions at both harvest times. Our data also demonstrate that the patterns of protein expression profiles are dose and time dependent. The majority of proteins with altered expression levels are involved in cell cycle control, cellular growth and proliferation, cell death and survival, cell-to-cell signaling and interaction. The IPA analyses indicate several important processes involved in responses to exposure to 48Ti ions. These include the proteosme/ubiquination, protein synthesis, post-translation modification, and lipid metabolism. The IPA analyses also indicate that exposure to 1 GeV/n 48Ti ions affects the development and function of hematological system, immune cell trafficking, including the cytoskeleton. Further, the IPA analyses strongly demonstrate that the NF-κB and MAPKs (ERKs, JNKs, and p38MAPK) pathways play an essential role in signal transduction after exposure to 1 GeV/n 48Ti ions. At an early time-point (1 week), the top networks identified by the IPA analyses are related to metabolic disease, lipid metabolism, small molecule biochemistry, and development disorder. In contrast, the top networks identified in samples collected at a late time-point (6 mos post-irradiation) by the IPA analyses are related to cancer, hematological disorders, and immunological diseases. In summary, the proteomic findings from our study provide a foundation to uncover compounds potentially be highly effective in radiation countermeasures.

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products.

Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611. Cytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication and delineated pIE611-dependent changes of the MCMV proteome. Our findings have fundamental implications for the interpretation of earlier studies on pIE3 functions and highlight the complex orchestration of MCMV gene regulation.

Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine

ABSTRACTThe development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90–110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.

Pathogenic Leptospira interrogans exoproteins are primarily involved in heterotrophic processes.

Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins.

Changes in glycoprotein expression between primary breast tumour and synchronous lymph node metastases or asynchronous distant metastases.

BackgroundBreast cancer is a very heterogeneous disease and some patients are cured by the surgical removal of the primary tumour whilst other patients suffer from metastasis and spreading of the disease, despite adjuvant therapy. A number of prognostic and treatment predictive factors have been identified such as tumour size, oestrogen (ER) and progesterone (PgR) receptor status, human epidermal growth factor receptor type 2 (HER2) status, histological grade, Ki67 and age. Lymph node involvement is also assessed during surgery to determine if the tumour has spread which requires dissection of the axilla and adjuvant treatment. The prognostic and treatment predictive factors assessing the nature of the tumour are all routinely based on the status of the primary tumour.ResultsWe have analysed a unique tumour set of fourteen primary breast cancer tumours with matched synchronous axillary lymph node metastases and a set of nine primary tumours with, later developed, matched distant metastases from different sites in the body. We used a pairwise tumour analysis (from the same individual) since the difference between the same tumour-type in different patients was greater. Glycopeptide capture was used in this study to selectively isolate and quantify N-linked glycopeptides from tumours mixtures and the captured glycopeptides were subjected to label-free quantitative tandem mass spectrometry analysis. Differentially expressed proteins between primary tumours and matched lymph node metastasis and distant metastasis were identified. Two of the top hits, ATPIF1 and tubulin β-chain were validated by immunohistochemistry to be differentially regulated.ConclusionsWe show that the expression of a large number of glycosylated proteins change between primary tumours and matched lymph node metastases and distant metastases, confirming that cancer cells undergo a molecular transformation during the spread to a secondary site. The proteins are part of important pathways such as cell adhesion, migration pathways and immune response giving insight into molecular changes needed for the tumour to spread. The large difference between primary tumours and lymph node and distant metastases also suggest that treatment should be based on the phenotype of the lymph node and distant metastases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9084-7) contains supplementary material, which is available to authorized users.

Summarization vs Peptide-Based Models in Label-Free Quantitative Proteomics: Performance, Pitfalls, and Data Analysis Guidelines.

Quantitative label-free mass spectrometry is increasingly used to analyze the proteomes of complex biological samples. However, the choice of appropriate data analysis methods remains a major challenge. We therefore provide a rigorous comparison between peptide-based models and peptide-summarization-based pipelines. We show that peptide-based models outperform summarization-based pipelines in terms of sensitivity, specificity, accuracy, and precision. We also demonstrate that the predefined FDR cutoffs for the detection of differentially regulated proteins can become problematic when differentially expressed (DE) proteins are highly abundant in one or more samples. Care should therefore be taken when data are interpreted from samples with spiked-in internal controls and from samples that contain a few very highly abundant proteins. We do, however, show that specific diagnostic plots can be used for assessing differentially expressed proteins and the overall quality of the obtained fold change estimates. Finally, our study also illustrates that imputation under the "missing by low abundance" assumption is beneficial for the detection of differential expression in proteins with low abundance, but it negatively affects moderately to highly abundant proteins. Hence, imputation strategies that are commonly implemented in standard proteomics software should be used with care.

Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells.

The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an “inclusion”. Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth.

Protein corona of nanoparticles: distinct proteins regulate the cellular uptake.

Understanding nanoparticle-protein interactions is a crucial issue in the development of targeted nanomaterial delivery. Besides unraveling the composition of the nanoparticle's protein coronas, distinct proteins thereof could control nanoparticle uptake into specific cell types. Here we differentially analyzed the protein corona composition on four polymeric differently functionalized nanoparticles by label-free quantitative mass spectrometry. Next, we correlated the relative abundance of identified proteins in the corona with enhanced or decreased cellular uptake of nanoparticles into human cancer and bone marrow stem cells to identify key candidates. Finally, we verified these candidate proteins by artificially decorating nanoparticles with individual proteins showing that nanoparticles precoated with the apolipoproteins ApoA4 or ApoC3 significantly decreased the cellular uptake, whereas precoating with ApoH increased the cellular uptake.

Quantitative tandem mass-spectrometry of skin tissue reveals putative psoriatic arthritis biomarkers.

BackgroundPsoriatic arthritis (PsA) is a distinct inflammatory arthritis occurring in 30% of psoriasis patients. There is a high prevalence of undiagnosed PsA in psoriasis patients; therefore, identifying soluble biomarkers for PsA could help in screening psoriasis patients for appropriate referral to a rheumatologist. Potential PsA biomarkers likely originate in sites of inflammation, such as the skin, and subsequently enter systemic circulation. Our goal was to identify candidate PsA biomarkers by comparing the proteome of skin biopsies obtained from patients with PsA to that from patients with psoriasis without PsA.MethodsSkin biopsies were obtained from involved and uninvolved skin of 10 PsA and 10 age/gender-matched psoriasis patients without PsA (PsC). Using strong cation exchange chromatography, followed by label-free quantitative tandem mass spectrometry, we characterized the proteomes of pooled skin samples. Extracted ion current intensities were used to calculate protein abundance ratios, and these were utilized to identify differentially regulated proteins.ResultsForty-seven proteins were elevated in PsA-derived skin compared to PsC-derived skin. Selected reaction monitoring assays were developed to quantify these potential PsA markers in individual skin samples, and 8 markers were confirmed in an independent sample set. ITGB5 and POSTN were measured in serum samples from 33 PsA and 15 PsC patients, using enzyme-linked immunosorbent assays. ITGB5 was significantly elevated in PsA serum (P < 0.01), and POSTN showed a trend. ITGB5 and POSTN correlated significantly in both patient groups (r = 0.472, P < 0.001).ConclusionProteomic analysis of PsA and PsC skin identified eight new candidate biomarkers. These markers need to be validated with a larger and independent cohort, in order to delineate their clinical utility in PsA patients. These proteins may also uncover unknown aspects of PsA pathobiology.Electronic supplementary materialThe online version of this article (doi:10.1186/1559-0275-12-1) contains supplementary material, which is available to authorized users.

Label-free quantitative mass spectrometry reveals a panel of differentially expressed proteins in colorectal cancer.

To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and DAVID databases were searched to identify the location and function of differential proteins that were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, and so on. Among the differentially expressed proteins, tropomyosin-3 (TPM3), endoplasmic reticulum resident protein 29 (ERp29), 18 kDa cationic antimicrobial protein (CAMP), and heat shock 70 kDa protein 8 (HSPA8) were verified to be upregulated in CRC tissue and seven cell lines through western blot analysis. Furthermore, the upregulation of TPM3, ERp29, CAMP, and HSPA8 was validated in 69 CRCs byimmunohistochemistry (IHC) analysis. Combination of TPM3, ERp29, CAMP, and HSPA8 can identify CRC from matched normal mucosal achieving an accuracy of 73.2% using IHC score. These results suggest that TPM3, ERp29, CAMP, and HSPA8 are great potential IHC diagnostic biomarkers for CRC.

Abstract 11072: Profiling of a Platelet Proteome Discriminates Between Subjects and the Effects of Antiplatelet Agents Alone and in Combination

Protein profiling of biological samples provides an integrated measurement of genetic, environmental and drug effects. We examined the response to a number of antiplatelet drugs alone and in combination on the human platelet secreteome following stimulation with thrombin. The drugs included a P2Y12 antagonist (PSB0739) and a PAR-1 antagonist (RWJ56110) at concentration (10nM) that blocked the platelet responses to their respective agonists, and aspirin (10microM). Thrombin (1U/ml) was used to stimulate aggregation and secretion of washed platelets despite the presence of the drugs. Platelets were obtained from healthy donors and drugs added in vitro. The proteome profile was determined by label-free quantitative mass spectrometry (Q Exactive and MaxQuant software). The profile was markedly different between individuals (n=3) but it was similar for the same individual over 3 separate samples obtained weeks apart. The addition of the antiplatelet agents showed a similar strong individual clustering of all proteins (&gt;1,000). Cluster analysis of the 270 proteins that changed with drug showed marked differences between drugs although there was no impact on platelet aggregation. Similarly, combinations of 2 or 3 drugs demonstrated distinctive clustering of changes in the protein profile. Functional ontology of the proteome showed marked differences between drugs with very distinctive patterns. Similarly, drugs differed clearly in their effects on discrete functional protein clusters, such as ‘coagulation proteins’. Our findings suggest that individuals have distinct platelet proteomes (‘proteome barcode’) that remain evident in the presence of drug. Despite this, antiplatelet drugs exhibit distinct effects on the platelet proteome, when applied alone and in combination. The profiling of the platelet proteome better discriminates the effects of antiplatelet drugs than is possible with platelet aggregation and may help tailor therapy more effectively.

Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Glioblastoma multiform (GBM) remains clinical indication with significant “unmet medical need”. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Mass Spectrometry and Imaging Analysis of Nanoparticle-Containing Vesicles Provide a Mechanistic Insight into Cellular Trafficking

Rational design of nanocarriers for drug delivery approaches requires an unbiased knowledge of uptake mechanisms and intracellular trafficking pathways. Here we dissected these processes using a quantitative proteomics approach. We isolated intracellular vesicles containing superparamagnetic iron oxide polystyrene nanoparticles and analyzed their protein composition by label-free quantitative mass spectrometry. The proteomic snapshot of organelle marker proteins revealed that an atypical macropinocytic-like mechanism mediated the entry of nanoparticles. We show that the entry mechanism is controlled by actin reorganization, atypical macropinocytic signaling, and ADP-ribosylation factor 1. Additionally, our proteomics data demonstrated a central role for multivesicular bodies and multilamellar lysosomes in trafficking and final nanoparticle storage. This was confirmed by confocal microscopy and cryo-TEM measurements. By quantitatively analyzing the protein composition of nanoparticle-containing vesicles, our study clearly defines the routes of nanoparticle entry, intracellular trafficking, and the proteomic milieu of a nanoparticle-containing vesicle.

Interactome analysis of the influenza A virus transcription/replication machinery identifies protein phosphatase 6 as a cellular factor required for efficient virus replication.

The negative-sense RNA genome of influenza A virus is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRP). The viral RdRP is an important host range determinant, indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Here, we employed affinity purification followed by mass spectrometry to identify cellular proteins that interact with the influenza A virus RdRP in infected human cells. We purified RdRPs using a recombinant influenza virus in which the PB2 subunit of the RdRP is fused to a Strep-tag. When this tagged subunit was purified from infected cells, copurifying proteins included the other RdRP subunits (PB1 and PA) and the viral nucleoprotein and neuraminidase, as well as 171 cellular proteins. Label-free quantitative mass spectrometry revealed that the most abundant of these host proteins were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, and mitochondrial and ribosomal proteins. Among the phosphatases, we identified three subunits of the cellular serine/threonine protein phosphatase 6 (PP6), including the catalytic subunit PPP6C and regulatory subunits PPP6R1 and PPP6R3. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and small interfering RNA (siRNA)-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. These results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP. Influenza A viruses are serious clinical and veterinary pathogens, causing substantial health and economic impacts. In addition to annual seasonal epidemics, occasional global pandemics occur when viral strains adapt to humans from other species. To replicate efficiently and cause disease, influenza viruses must interact with a large number of host factors. The reliance of the viral RNA-dependent RNA polymerase (RdRP) on host factors makes it a major host range determinant. This study describes and quantifies host proteins that interact, directly or indirectly, with a subunit of the RdRP. It increases our understanding of the role of host proteins in viral replication and identifies a large number of potential barriers to pandemic emergence. Identifying host factors allows their importance for viral replication to be tested. Here, we demonstrate a role for the cellular phosphatase PP6 in promoting viral replication, contributing to our emerging knowledge of regulatory phosphorylation in influenza virus biology.

Label-free protein quantification for plant Golgi protein localization and abundance.

The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (data-independent acquisition method using ion mobility separation known as LC-IMS-MS(E) [or HDMS(E)]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle.

Neuroproteomics in the auditory brainstem: candidate proteins for ultrafast and precise information processing.

In the mammalian auditory brainstem, the cochlear nuclear complex (CN) and the superior olivary complex (SOC) feature structural and functional specializations for ultrafast (<1 ms) and precise information processing. Their proteome, the basis for structure and function, has been rarely analyzed so far. Here we identified and quantified the protein profiles of three major auditory brainstem regions of adult rats, the CN, the SOC, and the inferior colliculus (IC). The rest of the brain served as a reference. Via label-free quantitative mass spectrometry and 2-D DIGE/MALDI-MS, we identified 584 and 297 proteins in the plasma membrane/synaptic vesicle proteome and the cytosolic proteome, respectively. 'Region-typical' proteins, i.e., those with higher abundance in one region than in the other three, were considered candidates for functional specializations. Key proteins were validated via Western blots and immunohistochemistry. Functional annotation clustering revealed an overrepresentation of neurofilament proteins among the CN+SOC-typical proteins. These are related to regulation of axon diameter and, thereby, conduction velocity. Interestingly, the sets of synapse-associated proteins differed between regions. For example, synaptotagmin-2 (Syt2), a Ca2+ sensor for fast exocytosis, was CN+SOC+IC-typical, whereas Syt1 was CN+SOC+IC-atypical. Together, our quantitative comparison of protein profiles has revealed several interesting candidate proteins for ultrafast and precise information processing.

The tegument protein pp65 of human cytomegalovirus acts as an optional scaffold protein that optimizes protein uploading into viral particles.

The mechanisms that lead to the tegumentation of herpesviral particles are only poorly defined. The phosphoprotein 65 (pp65) is the most abundant constituent of the virion tegument of human cytomegalovirus (HCMV). It is, however, nonessential for virion formation. This seeming discrepancy has not met with a satisfactory explanation regarding the role of pp65 in HCMV particle morphogenesis. Here, we addressed the question of how the overall tegument composition of the HCMV virion depended on pp65 and how the lack of pp65 influenced the packaging of particular tegument proteins. To investigate this, we analyzed the proteomes of pp65-positive (pp65pos) and pp65-negative (pp65neg) virions by label-free quantitative mass spectrometry and determined the relative abundances of tegument proteins. Surprisingly, only pUL35 was elevated in pp65neg virions. As the abundance of pUL35 in the HCMV tegument is low, it is unlikely that it replaced pp65 as a structural component in pp65neg virions. A subset of proteins, including the third most abundant tegument protein, pUL25, as well as pUL43, pUL45, and pUL71, were reduced in pp65neg or pp65low virions, indicating that the packaging of these proteins was related to pp65. The levels of tegument components, like pp28 and the capsid-associated tegument proteins pp150, pUL48, and pUL47, were unaffected by the lack of pp65. Our analyses demonstrate that deletion of pp65 is not compensated for by other viral proteins in the process of virion tegumentation. The results are concordant with a model of pp65 serving as an optional scaffold protein that facilitates protein upload into the outer tegument of HCMV particles. The assembly of the tegument of herpesviruses is only poorly understood. Particular proteins, like HCMV pp65, are abundant tegument constituents. pp65 is thus considered to play a major role in tegument assembly in the process of virion morphogenesis. We show here that deletion of the pp65 gene leads to reduced packaging of a subset of viral proteins, indicating that pp65 acts as an optional scaffold protein mediating protein upload into the tegument.

Mo1982 Potential Serum Biomarkers of Excessive Alcohol Use Discovered by Label-Free Quantitative Mass Spectrometry

Mo1982 Potential Serum Biomarkers of Excessive Alcohol Use Discovered by Label-Free Quantitative Mass Spectrometry