Label-free Protein Quantification Research Articles

1750P - Multi-level proteomics identifies FABP5 as a primary chemoresistance mediator in extensive-stage small cell lung cancer

BackgroundSmall cell lung cancer (SCLC), a highly invasive neuroendocrine tumor, accounts for approximately 15% of all new diagnosed lung cancer. Primary disease is highly sensitive to chemotherapy as well as radiotherapy, recurrences occur quickly, and less effective to subsequent therapies than the initial chemotherapy, the response rate of second-line therapy topotecan is only 22%. Drug resistance, is the main reason for failure of SCLC chemotherapy. Here, we quantitatively analyzed the proteomes of Cisplatin/Etoposide-resistant and -sensitive ES-SCLC patients from minute amounts of formalin-fixed and paraffin-embedded (FFPE) tissues. FABP5 was identified to be related with primary chemosensitivity, and may be an important mediator of Cisplatin/Etoposide-induced cell death. Methods51 tissue samples were collected from ES-SCLC patients who underwent Cisplatin and Etoposide chemotherapy in Zhe Jiang Cancer Hospital from January 2015 to November 2017. The 51 cases included 23 chemosensitive patients and 28 chemoresistant patients. System-wide quantitative proteomics approach was applied on the cohort of chemo-resistant and -sensitive tissues, and label free protein quantification was performed. Realtime-PCR was performed to assess the mRNA expression in H69 and multi-drug resistant H69AR cells. ResultsA total of 4,485 proteins were identified and quantified, more than 50% proteins were 10-70KD, Proteins with a fold difference greater than 1.5 (up/down) and a P value (t test) less than 0.05 were considered differentially expressed proteins, few overall proteome changes across the two groups. Comparison of the two-group found that FABP5 was significantly decreased in drug-resistant patients. Consistent with the protein level in tumor tissues, FABP5 mRNA was down-regulated in H69AR cells. FABP5 expression strongly correlated with decreased disease-free survival and overall survival in ES-SCLC patients. ConclusionsThese data revealed FABP5 may serve as a potential biomarker for primary chemoresistance and prognostic factor for patients with ES-SCLC, which need further research. Legal entity responsible for the studyYamei Chen. FundingNational Natural Science Foundation of China (grant numbers 81672972). DisclosureAll authors have declared no conflicts of interest.

Plasma membrane proteome analyses of Arabidopsis thaliana suspension-cultured cells during cold or ABA treatment: Relationship with freezing tolerance and growth phase

Cold acclimation (CA) and abscisic acid (ABA) treatments affected freezing tolerance of Arabidopsis thaliana suspension-cultured cells: cells at the lag and log phases increased their freezing tolerance but cells at the stationery phase rather decreased after the treatments. To further characterize the differential responses of the cells to these treatments depending on growth phase, plasma membrane (PM) proteome were analyzed using label-free protein quantification technology. Abundance of 841 proteins changed during the progression of growth phase, CA and/or ABA treatment. Among them, 392 proteins changed in their abundance in cells during growth phase progression and were categorized into various functional groups, suggesting changes in physiological characteristics of the PM depending on the growth phase. Comparison of CA- and ABA-responsive proteins indicated that ABA is one of the important signals for PM proteome changes in response to CA, but multiple signals are required for the response of PM proteins to CA. Involvement of ABA in the CA process diminished with the progression of growth phase. Taken together, the results suggest that dynamic alterations of the PM proteome with the progression of growth phase influence the PM proteome responses to CA and ABA, which may effect the difference in freezing tolerance capability. SignificanceAfter cold acclimation (CA) or abscisic acid treatment (ABA), Arabidopsis thaliana suspension-cultured cells (T87 line) exhibited freezing tolerance capability dependent on the cell growth phase. However, whether the plasma membrane (PM) proteome profile differs among growth phases and the differences are associated with growth-phase-dependent freezing tolerance have not been elucidated. In the present study, PM proteomics was conducted in association with CA and ABA treatment of Arabidopsis suspension-cultured cells at three growth phases. Characteristics of the PM proteome changed considerably with progression of the growth phase and ABA was indicated to be an important signal for PM protein changes during CA. The results also revealed that multiple signals are required to complete the response of PM proteins to CA. Therefore, dynamic alterations of the PM proteome with the advance of the growth phase influence the responses of PM proteome to CA and ABA, which may result in the differences in freezing tolerance of the cultured cells.

Royal jelly-derived proteins enhance proliferation and migration of human epidermal keratinocytes in an in vitro scratch wound model

BackgroundSkin injury is inevitable in daily life. In recent years, with the increasing morbidity of diseases such as diabetes and metabolic disorders, chronic wounds have become a considerable challenge in clinical practice. Royal jelly, reported to have multifarious biological and physiological properties, has been used as a remedy for a variety of wounds since ancient times. However, the active components and mechanisms underlying the wound-healing properties of royal jelly are still largely unknown.MethodsWater-soluble proteins of royal jelly were fractionated and investigated for the proliferative and migratory effects on human epidermal keratinocytes (HaCaT) in an in vitro wound healing model. The proteins present in bioactive fractions were characterised and quantified using Label-free protein quantification method. The potential functions of these proteins in biological systems were further analysed using bioinformatic tools.ResultsA protein fraction, mainly containing major royal jelly proteins 2 (MRJP2), MRJP3 and MRJP7, stimulated proliferative and migratory activities in HaCaT cells without visible cytotoxicity. It exerted the greatest effects on the growth of HaCaT cells in the first 48 h. Furthermore, when treated with this protein fraction, the closure rates of the in vitro scratch wound were significantly increased. Functional analysis indicated that MRJP2, MRJP3 and MRJP7 were associated with carbohydrate transport and metabolism.ConclusionsWe fractionated the water-soluble proteins of royal jelly and identified one fraction (Fraction 2) that induced both proliferative and migratory effects on a human epidermal keratinocyte cell line. Major royal jelly proteins (MRJP2, MRJP3 and/or MRJP7) were speculated to possess potential wound-healing bioactivity. This is the first report that royal jelly may improve wound closure via MRJP-induced cellular proliferation and migration. These proteins may be valuable lead compounds for the development of novel wound healing medications. Our findings would facilitate better understanding of the wound repair mechanisms of royal jelly.

More than a Toxin: Protein Inventory of Clostridium tetani Toxoid Vaccines.

Clostridium tetani is the etiological agent of tetanus, a life-threatening bacterial infection. The most efficient protection strategy against tetanus is a vaccination with the C. tetani neurotoxin, which is inactivated by formaldehyde-crosslinking. Since we assumed that besides the tetanus toxin, other proteins of C. tetani may also be present in toxoid preparations, we analyzed commercially available vaccines from different countries in respect to their protein content using mass spectrometry. In total 991 proteins could be identified in all five analyzed vaccines, 206 proteins were common in all analyzed vaccines and 54 proteins from the 206 proteins were potential antigens. The additionally present proteins may contribute at least partially to protection against C. tetani infection by supporting the function of the vaccine against the devastating effects of the tetanus toxin indirectly. Two different label-free protein quantification methods were applied for an estimation of protein contents. Similar results were obtained with a Total Protein Approach (TPA)-based method and Protein Discoverer 2.2 software package based on the minora algorithm. Depending on the tetanus toxoid vaccine and the quantification method used, tetanus neurotoxin contributes between 14 and 76 % to the total C. tetani protein content and varying numbers of other C. tetani proteins were detected.

Label-free absolute protein quantification with data-independent acquisition

Despite data-independent acquisition (DIA) has been increasingly used for relative protein quantification, DIA-based label-free absolute quantification method has not been fully established. Here we present a novel DIA method using the TPA algorithm (DIA-TPA) for the absolute quantification of protein expressions in human liver microsomal and S9 samples. To validate this method, both data-dependent acquisition (DDA) and DIA experiments were conducted on 36 individual human liver microsome and S9 samples. The MS2-based DIA-TPA was able to quantify approximately twice as many proteins as the MS1-based DDA-TPA method, whereas protein concentrations determined by the two approaches were comparable. To evaluate the accuracy of the DIA-TPA method, we absolutely quantified carboxylesterase 1 concentrations in human liver S9 fractions using an established SILAC internal standard-based proteomic assay; the SILAC results were consistent with those obtained from DIA-TPA analysis. Finally, we employed a unique algorithm in DIA-TPA to distribute the MS signals from shared peptides to individual proteins or isoforms and successfully applied the method to the absolute quantification of several drug-metabolizing enzymes in human liver microsomes. In sum, the DIA-TPA method not only can absolutely quantify entire proteomes and specific proteins, but also has the capability quantifying proteins with shared peptides. SignificanceData independent acquisition (DIA) has emerged as a powerful approach for relative protein quantification at the whole proteome level. However, DIA-based label-free absolute protein quantification (APQ) method has not been fully established. In the present study, we present a novel DIA-based label-free APQ approach, named DIA-TPA, with the capability absolutely quantifying proteins with shared peptides. The method was validated by comparing the quantification results of DIA-TPA with that obtained from stable isotope-labeled internal standard-based proteomic assays.

Spectral Clustering Improves Label-Free Quantification of Low-Abundant Proteins.

Label-free quantification has become a common-practice in many mass spectrometry-based proteomics experiments. In recent years, we and others have shown that spectral clustering can considerably improve the analysis of (primarily large-scale) proteomics data sets. Here we show that spectral clustering can be used to infer additional peptide-spectrum matches and improve the quality of label-free quantitative proteomics data in data sets also containing only tens of MS runs. We analyzed four well-known public benchmark data sets that represent different experimental settings using spectral counting and peak intensity based label-free quantification. In both approaches, the additionally inferred peptide-spectrum matches through our spectra-cluster algorithm improved the detectability of low abundant proteins while increasing the accuracy of the derived quantitative data, without increasing the data sets’ noise. Additionally, we developed a Proteome Discoverer node for our spectra-cluster algorithm which allows anyone to rebuild our proposed pipeline using the free version of Proteome Discoverer.

Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus.

Neutrophil Extracellular Traps (NETs) are implicated in the development of auto-immunity in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) through the externalization of intracellular neoepitopes e.g., dsDNA and nuclear proteins in SLE and citrullinated peptides in RA. The aim of this work was to use quantitative proteomics to identify and measure NET proteins produced by neutrophils from healthy controls, and from patients with RA and SLE to determine if NETs can be differentially-generated to expose different sets of neoepitopes. Ultra-pure neutrophils (>99%) from healthy individuals (n = 3) and patients with RA or SLE (n = 6 each) were incubated ± PMA (50 nM, PKC super-activator) or A23187 (3.8 μM, calcium ionophore) for 4 h. NETs were liberated by nuclease digestion and concentrated onto Strataclean beads prior to on-bead digestion with trypsin. Data-dependent LC-MS/MS analyses were conducted on a QExactive HF quadrupole-Orbitrap mass spectrometer, and label-free protein quantification was carried out using Progenesis QI. PMA-induced NETs were decorated with annexins, azurocidin and histone H3, whereas A23187-induced NETs were decorated with granule proteins including CAMP/LL37, CRISP3, lipocalin and MMP8, histones H1.0, H1.4, and H1.5, interleukin-8, protein-arginine deiminase-4 (PADI4), and α-enolase. Four proteins were significantly different between PMA-NETs from RA and SLE neutrophils (p < 0.05): RNASE2 was higher in RA, whereas MPO, leukocyte elastase inhibitor and thymidine phosphorylase were higher in SLE. For A23187-NETs, six NET proteins were higher in RA (p < 0.05), including CAMP/LL37, CRISP3, interleukin-8, MMP8; Thirteen proteins were higher in SLE, including histones H1.0, H2B, and H4. This work provides the first, direct comparison of NOX2-dependent (PMA) and NOX2-independent (A23187) NETs using quantitative proteomics, and the first direct comparison of RA and SLE NETs using quantitative proteomics. We show that it is the nature of the stimulant rather than neutrophil physiology that determines NET protein profiles in disease, since stimulation of NETosis in either a NOX2-dependent or a NOX2-independent manner generates broadly similar NET proteins irrespective of the disease background. We also use our proteomics pipeline to identify an extensive range of post-translationally modified proteins in RA and SLE, including histones and granule proteins, many of which are known targets of auto-antibodies in each disease.

Fast Proteome Identification and Quantification from Data-Dependent Acquisition–Tandem Mass Spectrometry (DDA MS/MS) Using Free Software Tools

The identification of nearly all proteins in a biological system using data-dependent acquisition (DDA) tandem mass spectrometry has become routine for organisms with relatively small genomes such as bacteria and yeast. Still, the quantification of the identified proteins may be a complex process and often requires multiple different software packages. In this protocol, I describe a flexible strategy for the identification and label-free quantification of proteins from bottom-up proteomics experiments. This method can be used to quantify all the detectable proteins in any DDA dataset collected with high-resolution precursor scans and may be used to quantify proteome remodeling in response to drug treatment or a gene knockout. Notably, the method is statistically rigorous, uses the latest and fastest freely-available software, and the entire protocol can be completed in a few hours with a small number of data files from the analysis of yeast.

Single-cell proteomics in complex tissues using microprobe capillary electrophoresis mass spectrometry.

Direct measurement of proteins produced by single cells promises to expand our understanding of molecular cell-to-cell differences (heterogeneity) and their contribution to normal and impaired development. High-resolution mass spectrometry (HRMS) is the modern technology of choice for the label-free identification and quantification of proteins, albeit usually in large populations of cells. Recent advances in microscale sample collection and processing, separation, and ionization have extended this powerful technology to single cells. This chapter describes a protocol based on microprobe capillary electrophoresis (CE) HRMS to enable the direct proteomic profiling of single cells embedded in complex tissues without the requirement for dissociation or whole-cell dissection. We here demonstrate the technology for identified individual cells in early developing embryos of Xenopus laevis and zebrafish as well as electrophysiologically identified single neurons in physiologically active brain slices from the mouse substantia nigra. Instructions are provided step-by-step to identify single cells using physiological or morphological cues, collect the content of the cells using microfabricated capillaries, and perform bottom-up proteomics using a custom-built CE electrospray ionization (ESI) mass spectrometer equipped with a quadrupole time-of-flight or orbitrap mass analyzer. Results obtained by this approach have revealed previously unknown differences between the proteomic state of embryonic cells and neurons. The data from single-cell proteomics by microprobe CE-ESI-HRMS complements those from single-cell transcriptomics, thereby opening exciting potentials to deepen our knowledge of molecular mechanisms governing cell and developmental processes.

Label-free protein quantification after ultrafast digestion of complex proteomes using ultrasonic energy and immobilized-trypsin magnetic nanoparticles

The ultimate high-throughput, high robustness and easy-to-handling sample treatment method for label-free shotgun proteomics is presented in this work. It is based on joining the effectiveness of immobilized trypsin at the nanoscale level with the latest technology to deliver ultrasonic energy. The new method can be used to reduce sample preparation time comprising the steps of reduction, alkylation and digestion time to just 15 min without compromising shotgun label-free protein quantification. It is demonstrated that trypsin immobilized at the nano-scale performs better than the commercially available counterpart macroparticles. Considering the current advances in (i) ultrasonic energy delivery that allows 96 samples to be treated at once in 30 min, and (ii) chromatography and mass spectrometry for shotgun proteomics, that allow to analyze complex proteomes in 5 min, we envision this methodology as the universal one to digest complex proteomes as it allows to profile quantitatively more than 200 samples per day.

LFAQ: Toward Unbiased Label-Free Absolute Protein Quantification by Predicting Peptide Quantitative Factors.

Mass spectrometry (MS) has become a predominant choice for large-scale absolute protein quantification, but its quantification accuracy still has substantial room for improvement. A crucial issue is the bias between the peptide MS intensity and the actual peptide abundance, i.e., the fact that peptides with equal abundance may have different MS intensities. This bias is mainly caused by the diverse physicochemical properties of peptides. Here, we propose an algorithm for label-free absolute protein quantification, LFAQ, which can correct the biased MS intensities by using the predicted peptide quantitative factors for all identified peptides. When validated on data sets produced by different MS instruments and data acquisition modes, LFAQ presented accuracy and precision superior to those of existing methods. In particular, it reduced the quantification error by an average of 46% for low-abundance proteins. The advantages of LFAQ were further confirmed using the data from published papers.

The Complexome of Dehalococcoides mccartyi Reveals Its Organohalide Respiration-Complex Is Modular.

Dehalococcoides mccartyi strain CBDB1 is a slow growing strictly anaerobic microorganism dependent on halogenated compounds as terminal electron acceptor for anaerobic respiration. Indications have been described that the membrane-bound proteinaceous organohalide respiration complex of strain CBDB1 is functional without quinone-mediated electron transfer. We here study this multi-subunit protein complex in depth in regard to participating protein subunits and interactions between the subunits using blue native gel electrophoresis coupled to mass spectrometric label-free protein quantification. Applying three different solubilization modes to detach the respiration complex from the membrane we describe different solubilization snapshots of the organohalide respiration complex. The results demonstrate the existence of a two-subunit hydrogenase module loosely binding to the rest of the complex, tight binding of the subunit HupX to OmeA and OmeB, predicted to be the two subunits of a molybdopterin-binding redox subcomplex, to form a second module, and the presence of two distinct reductive dehalogenase module variants with different sizes. In our data we obtained biochemical evidence for the specificity between a reductive dehalogenase RdhA (CbdbA80) and its membrane anchor protein RdhB (CbdbB3). We also observed weak interactions between the reductive dehalogenase and the hydrogenase module suggesting a not yet recognized contact surface between these two modules. Especially an interaction between the two integral membrane subunits OmeB and RdhB seems to promote the integrity of the complex. With the different solubilization strengths we observe successive disintegration of the complex into its subunits. The observed architecture would allow the association of different reductive dehalogenase modules RdhA/RdhB with the other two protein complex modules when the strain is growing on different electron acceptors. In the search for other respiratory complexes in strain CBDB1 the remarkable result is not the detection of a standard ATPase but the absence of any other abundant membrane complex although an 11-subunit version of complex I (Nuo) is encoded in the genome.

MSE for Label-Free Absolute Protein Quantification in Complex Proteomes.

Label-free peptide quantification is a promising approach for the large-scale characterization of proteome dynamics at low cost. Here, we describe a method for absolute label-free quantification using an untargeted approach for peptide fragmentation referred to as MSE. We show that spiked external standards provide sufficient accuracy for the quantification of proteins in complex samples resulting in similar protein quantification results as spectral counting. As an advantage, label-free quantification also works for small numbers of samples whereas spectral counting requires large datasets to result in a similar robustness. The sensitivity of protein identification increases significantly when ion mobility separation is included in addition to the standard LC-MS setup in the analysis workflow. Ion mobility decreases sample complexity and serves as an additional separation criterion to align a parent ion with its product ions after MSE fragmentation. As a drawback, quantification of high abundance proteins becomes inaccurate because of detector saturation. We describe here a suitable workflow to achieve good sensitivity for protein quantification and give initial guidance on data interpretation. To achieve good identification and quantification accuracy, the protein amount loaded onto the column should not exceed 400-600ng. In a dynamic range window of 3-4 orders of magnitude, robust quantification can be obtained with complex samples comprising up to 2000-3000 proteins.

Ultrasonic-based membrane aided sample preparation of urine proteomes

A new ultrafast ultrasonic-based method for shotgun proteomics as well as label-free protein quantification in urine samples is developed. The method first separates the urine proteins using nitrocellulose-based membranes and then proteins are in-membrane digested using trypsin. The enzymatic digestion process is accelerated from overnight to four minutes using a sonoreactor ultrasonic device. Overall, the sample treatment pipeline comprising protein separation, digestion and identification is done in just 3h. The process is assessed using urine of healthy volunteers. The method shows that male can be differentiated from female using the protein content of urine in a fast, easy and straightforward way. 232 and 226 proteins are identified in urine of male and female, respectively. From this, 162 are common to both genders, whilst 70 are unique to male and 64 to female. From the 162 common proteins, 13 are present at levels statistically different (p < 0.05). The method matches the analytical minimalism concept as outlined by Halls, as each stage of this analysis is evaluated to minimize the time, cost, sample requirement, reagent consumption, energy requirements and production of waste products.

Cerebrospinal fluid markers to distinguish bacterial meningitis from cerebral malaria in children.

Background. Few hospitals in high malaria endemic countries in Africa have the diagnostic capacity for clinically distinguishing acute bacterial meningitis (ABM) from cerebral malaria (CM). As a result, empirical use of antibiotics is necessary. A biochemical marker of ABM would facilitate precise clinical diagnosis and management of these infections and enable rational use of antibiotics. Methods. We used label-free protein quantification by mass spectrometry to identify cerebrospinal fluid (CSF) markers that distinguish ABM (n=37) from CM (n=22) in Kenyan children. Fold change (FC) and false discovery rates (FDR) were used to identify differentially expressed proteins. Subsequently, potential biomarkers were assessed for their ability to discriminate between ABM and CM using receiver operating characteristic (ROC) curves. Results. The host CSF proteome response to ABM ( Haemophilusinfluenza and Streptococcuspneumoniae) is significantly different to CM. Fifty two proteins were differentially expressed (FDR<0.01, Log FC≥2), of which 83% (43/52) were upregulated in ABM compared to CM. Myeloperoxidase and lactotransferrin were present in 37 (100%) and 36 (97%) of ABM cases, respectively, but absent in CM (n=22). Area under the ROC curve (AUC), sensitivity, and specificity were assessed for myeloperoxidase (1, 1, and 1; 95% CI, 1-1) and lactotransferrin (0.98, 0.97, and 1; 95% CI, 0.96-1). Conclusion. Myeloperoxidase and lactotransferrin have a high potential to distinguish ABM from CM and thereby improve clinical management. Their validation requires a larger cohort of samples that includes other bacterial aetiologies of ABM.

Cerebrospinal fluid markers to distinguish bacterial meningitis from cerebral malaria in children

Background . Few hospitals in high malaria endemic countries in Africa have the diagnostic capacity for clinically distinguishing acute bacterial meningitis (ABM) from cerebral malaria (CM). As a result, empirical use of antibiotics is necessary. A biochemical marker of ABM would facilitate precise clinical diagnosis and management of these infections and enable rational use of antibiotics. Methods. We used label-free protein quantification by mass spectrometry to identify cerebrospinal fluid (CSF) markers that distinguish ABM (n=37) from CM (n=22) in Kenyan children. Fold change (FC) and false discovery rates (FDR) were used to identify differentially expressed proteins. Subsequently, potential biomarkers were assessed for their ability to discriminate between ABM and CM using receiver operating characteristic (ROC) curves. Results. The host CSF proteome response to ABM ( Haemophilus influenza and Streptococcus pneumoniae) is significantly different to CM. Fifty two proteins were differentially expressed (FDR<0.01, Log FC≥2), of which 83% (43/52) were upregulated in ABM compared to CM. Myeloperoxidase and lactotransferrin were present in 37 (100%) and 36 (97%) of ABM cases, respectively, but absent in CM (n=22). Area under the ROC curve (AUC), sensitivity, and specificity were assessed for myeloperoxidase (1, 1, and 1; 95% CI, 1-1) and lactotransferrin (0.98, 0.97, and 1; 95% CI, 0.96-1). Conclusion. Myeloperoxidase and lactotransferrin have a high potential to distinguish ABM from CM and thereby improve clinical management. Their validation requires a larger cohort of samples that includes other bacterial aetiologies of ABM.

Cerebrospinal fluid markers to distinguish bacterial meningitis from cerebral malaria in children

Background. Few hospitals in high malaria endemic countries in Africa have the diagnostic capacity for clinically distinguishing acute bacterial meningitis (ABM) from cerebral malaria (CM). As a result, empirical use of antibiotics is necessary. A biochemical marker of ABM would facilitate precise clinical diagnosis and management of these infections and enable rational use of antibiotics. Methods. We used label-free protein quantification by mass spectrometry to identify cerebrospinal fluid (CSF) markers that distinguish ABM (n=37) from CM (n=22) in Kenyan children. Fold change (FC) and false discovery rates (FDR) were used to identify differentially expressed proteins. Subsequently, potential biomarkers were assessed for their ability to discriminate between ABM and CM using receiver operating characteristic (ROC) curves. Results. The host CSF proteome response to ABM (Haemophilus influenza and Streptococcus pneumoniae) is significantly different to CM. Fifty two proteins were differentially expressed (FDR&lt;0.01, Log FC≥2), of which 83% (43/52) were upregulated in ABM compared to CM. Myeloperoxidase and lactotransferrin were present in 37 (100%) and 36 (97%) of ABM cases, respectively, but absent in CM (n=22). Area under the ROC curve (AUC), sensitivity, and specificity were assessed for myeloperoxidase (1, 1, and 1; 95% CI, 1-1) and lactotransferrin (0.98, 0.97, and 1; 95% CI, 0.96-1). Conclusion. Myeloperoxidase and lactotransferrin have a high potential to distinguish ABM from CM and thereby improve clinical management. Their validation requires a larger cohort of samples that includes other bacterial aetiologies of ABM.

PROTEOMICS IDENTIFICATION OF NOVEL CEREBROSPINAL FLUID BIOMARKER CANDIDATES OF DEMENTIA WITH LEWY BODIES

Cerebrospinal fluid (CSF) is considered an optimal source for the discovery of in vivo biomarkers for Dementia with Lewy bodies (DLB). Thus far, no reliable biomarker with both high sensitivity and specificity is available for DLB. CSF biomarkers could improve the early diagnostic accuracy, monitor disease progression and treatment response. We set out a proteomics study to identify novel CSF biomarker candidates for DLB. An in-depth proteomics workflow involving immune-depletion of high-abundant proteins, mono-dimensional SDS-PAGE in conjunction with nanoLC-MS/MS-based proteomics, database searching (MaxQuant) and label-free protein quantification was applied on CSF of 20 probable DLB patients and age and gender matched individuals with subjective cognitive decline (SCD, n=20). DLB patients had an abnormal (123)I-FP-CIT SPECT scan and normal CSF values of Aβ42, total tau and phosphorylated tau. Candidate biomarkers were selected based on the following criteria: (1) p<0.05, fold change>1.2, median normalized spectral count ≥2; (2) ≥50% separation between the two groups; and (3) identified peptide sequences covering ≥20% of the protein. Fifty-seven out of 2100 identified proteins were differentially expressed in DLB patients compared to SCD (p<0.05). 35 proteins were down-regulated and 22 proteins were up-regulated in DLB compared to SCD. We selected three proteins for validation by ELISA in a cohort including DLB, AD, Parkinson's disease (PD) and SCD. This validation is currently in progress. Our proteomics analysis of CSF identified several novel potential candidate biomarkers for DLB. Further validation of prioritized candidates in larger cohorts should delineate their potential as early diagnostic and/or progression biomarkers for DLB.

Label-free quantification of meat proteins for evaluation of species composition of processed meat products

This study evaluated the application of label-free, relative quantification of highly processed meat proteins to authenticate processed meat products. Quantitation was based on proteins for which heat-stable and species-specific peptide markers had been detected. Using nano-LC-QTOF-MS/MS, 20 new, heat-stable peptide markers unique to chicken, duck and goose were identified, with significant scores. The method enabled detection of 1%(w/w) of chicken and 1%(w/w) pork in a mixture of the meat of three species, as well as 0.8%(w/w) beef proteins in commercial poultry frankfurters. The proteins’ abundance was computed using a spectral matching technique, termed the absolute protein expression (APEX) proteomics tool. This method includes a correction factor for each protein, based on the peptide MS detection probabilities, which are influenced by the physicochemical properties of the peptide. Considerable differences in abundance of myofibrillar and sarcoplasmic proteins were observed between samples and illegal proportions of ingredients were discovered.

Quantification of egg proteome changes during fertilization in sterlet Acipenser ruthenus

Eggs of sterlet are discharged outside into ambient aquatic environment where egg activation and fertilization occur. Effects of different activation media including freshwater and clay suspension on protein abundances of egg were quantified in sterlet Acipenser ruthenus. In-gel digestion and high resolution mass spectrometry were used for label-free protein quantification in the eggs of five females. No significant (p > 0.05) difference was found between protein abundances in eggs activated with different media. However, results showed significant (p < 0.05, fold change ≥2) reduction in the abundances of nine proteins including six glycoproteins, enolase and heat shock protein in activated groups compared to freshly ovulated eggs as control. The fact that abundance of proteasome subunit alpha significantly reduced only in eggs which were activated by clay suspension suggests that activation medium can somehow intervene with protein regulation during fertilization. In conclusion, external fertilization in sturgeon egg is accompanied by huge release of proteins into the external environment that may participate in the construction of a transient microenvironment around egg for attraction and protection of spermatozoa to ensure ensuing fertilization. Data are available via ProteomeXchange with identifier PXD006232.