Nanoflow Liquid Chromatography Research Articles

High-resolution droplet-based fractionation of nano-LC separations onto microarrays for MALDI-MS analysis.

We present a robust droplet-based device, which enables the fractionation of ultralow flow rate nanoflow liquid chromatography (nano-LC) eluate streams at high frequencies and high peak resolution. This is achieved by directly interfacing the separation column to a micro T-junction, where the eluate stream is compartmentalized into picoliter droplets. This immediate compartmentalization prevents peak dispersion during eluate transport and conserves the chromatographic performance. Subsequently, nanoliter eluate fractions are collected at a rate of one fraction per second on a high-density microarray to retain the separation with high temporal resolution. Chromatographic separations of up to 45 min runtime can thus be archived on a single microarray possessing 2700 sample spots. The performance of this device is demonstrated by fractionating the separation of a tryptic digest of a known protein mixture onto the microarray chip and subsequently analyzing the sample archive using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Resulting peak widths are found to be significantly reduced compared to standard continuous flow spotting technologies as well as in comparison to a conventional nano-LC-electrospray ionization-mass spectrometry interface. Moreover, we demonstrate the advantage of our high-definition nanofractionation device by applying two different MALDI matrices to all collected fractions in an alternating fashion. Since the information that is obtained from a MALDI-MS measurement depends on the choice of MALDI matrix, we can extract complementary information from neighboring spots containing almost identical composition but different matrices.

Combinational treatment of colon cancer cells with bioactive food components (1147.2)

Using comparative proteomic analysis, we investigated the synergistic suppressive activity of natural components on the metastasis of colorectal cancer (CRC) cells. The isogenic SW480 and SW620 colon adenocarcinoma cell lines were compared to identify proteins overexpressed in metastatic SW620, which demonstrated altered expression levels following treatment with bioactive food components from natural sources and/or chemotherapeutic agents. SW620 cells were treated with three natural components, namely, curcumin, luteolin, and ginsenoside 20(S)‐Rg3 and three chemotherapeutic agents, 5‐fluorouracil, sorafenib, and oxaliplatin, which were selected by their cytotoxicity to SW620 cells. Comparative proteomic datasets were generated using linear ion‐trap mass spectrometry combined with nano‐flow ultra performance liquid chromatography. MS data files were searched using the SEQUEST algorithm. Significant overexpression of structural proteins, such as neurofilaments and histone proteins, was observed in untreated SW620 compared to untreated SW480 cells. Interestingly, treatment with curcumin or ginsenoside 20(S)‐Rg3 suppressed the overexpression more effectively than either 5‐FU or oxaliplatin, which are drugs currently used as primary therapy in the treatment of colorectal cancer. In addition, the overexpression of histone proteins in SW620 cells was down‐regulated by the co‐treatment of curcumin and oxaliplatin, which demonstrated synergistic cytotoxicity via combinational treatment. It has been known that chemical modification (e.g. acetylation) of histones is one of the major features of cancer metastasis. However, based on the current proteomic analysis, increased expression of histone proteins is also associated with the development of CRC metastasis. Furthermore, our data suggests that curcumin co‐treatment represents a promising adjuvant to standard oxaliplatin therapy for the suppression of metastasis in colorectal adenocarcinoma.

A Remodeled Protein Arginine Methyltransferase 1 (PRMT1) Generates Symmetric Dimethylarginine

Protein arginine methylation is emerging as a significant post-translational modification involved in various cell processes and human diseases. As the major arginine methylation enzyme, protein arginine methyltransferase 1 (PRMT1) strictly generates monomethylarginine and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA). The two types of dimethylarginines can lead to distinct biological outputs, as highlighted in the PRMT-dependent epigenetic control of transcription. However, it remains unclear how PRMT1 product specificity is regulated. We discovered that a single amino acid mutation (Met-48 to Phe) in the PRMT1 active site enables PRMT1 to generate both ADMA and SDMA. Due to the limited amount of SDMA formed, we carried out quantum mechanical calculations to determine the free energies of activation of ADMA and SDMA synthesis. Our results indicate that the higher energy barrier of SDMA formation (ΔΔG(‡) = 3.2 kcal/mol as compared with ADMA) may explain the small amount of SDMA generated by M48F-PRMT1. Our study reveals unique energetic challenges for SDMA-forming methyltransferases and highlights the exquisite control of product formation by active site residues in the PRMTs.

Discovery of Novel Disease-specific and Membrane-associated Candidate Markers in a Mouse Model of Multiple Sclerosis

Multiple sclerosis is a chronic demyelinating disorder characterized by the infiltration of auto-reactive immune cells from the periphery into the central nervous system resulting in axonal injury and neuronal cell death. Experimental autoimmune encephalomyelitis represents the best characterized animal model as common clinical, histological, and immunological features are recapitulated. A label-free mass spectrometric proteomics approach was used to detect differences in protein abundance within specific fractions of disease-affected tissues including the soluble lysate derived from the spinal cord and membrane protein-enriched peripheral blood mononuclear cells. Tissues were harvested from actively induced experimental autoimmune encephalomyelitis mice and sham-induced ("vehicle" control) counterparts at the disease peak followed by subsequent analysis by nanoflow liquid chromatography tandem mass spectrometry. Relative protein quantitation was performed using both intensity- and fragmentation-based approaches. After statistical evaluation of the data, over 500 and 250 differentially abundant proteins were identified in the spinal cord and peripheral blood mononuclear cell data sets, respectively. More than half of these observations have not previously been linked to the disease. The biological significance of all candidate disease markers has been elucidated through rigorous literature searches, pathway analysis, and validation studies. Results from comprehensive targeted mass spectrometry analyses have confirmed the differential abundance of ∼ 200 candidate markers (≥ twofold dysregulated expression) at a 70% success rate. This study is, to our knowledge, the first to examine the cell-surface proteome of peripheral blood mononuclear cells in experimental autoimmune encephalomyelitis. These data provide a unique mechanistic insight into the dynamics of peripheral immune cell infiltration into CNS-privileged sites at a molecular level and has identified several candidate markers, which represent promising targets for future multiple sclerosis therapies. The mass spectrometry proteomics data associated with this manuscript have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000255.

Nano-LC/MALDI-MS Using a Column-Integrated Spotting Probe for Analysis of Complex Biomolecule Samples

Nanoflow liquid chromatography (nano-LC) is an essential technique for highly sensitive analysis of complex biological samples, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is advantageous for rapid identification of proteins and in-depth analysis of post-translational modifications (PTMs). A combination of nano-LC and MALDI-MS (nano-LC/MALDI-MS) is useful for highly sensitive and detailed analysis in life sciences. However, the existing system does not fully utilize the advantages of each technique, especially in the interface of eluate transfer from nano-LC to a MALDI plate. To effectively combine nano-LC with MALDI-MS, we integrated a nano-LC column and a deposition probe for the first time (column probe) and incorporated it into a nano-LC/MALDI-MS system. Spotting nanoliter eluate droplets directly from the column onto the MALDI plate prevents postcolumn diffusion and preserves the chromatographic resolution. A DHB prespotted plate was prepared to suit the fabricated column probe to concentrate the droplets of nano-LC eluate. The performance of the advanced nano-LC/MALDI-MS system was substantiated by analyzing protein digests. When the system was coupled with multidimensional liquid chromatography (MDLC), trace amounts of glycopeptides that spiked into complex samples were successfully detected. Thus, a nano-LC/MALDI-MS direct-spotting system that eliminates postcolumn diffusion was constructed, and the efficacy of the system was demonstrated through highly sensitive analysis of the protein digests or spiked glycopeptides.

Qualitative and Quantitative Characterization of Plasma Proteins When Incorporating Traveling Wave Ion Mobility into a Liquid Chromatography–Mass Spectrometry Workflow for Biomarker Discovery: Use of Product Ion Quantitation As an Alternative Data Analysis Tool for Label Free Quantitation

Discovery of protein biomarkers in clinical samples necessitates significant prefractionation prior to liquid chromatography–mass spectrometry (LC–MS) analysis. Integrating traveling wave ion mobility spectrometry (TWIMS) enables in-line gas phase separation which when coupled with nanoflow liquid chromatography and data independent acquisition tandem mass spectrometry, confers significant advantages to the discovery of protein biomarkers by improving separation and inherent sensitivity. Incorporation of TWIMS leads to a packet of concentrated ions which ultimately provides a significant improvement in sensitivity. As a consequence of ion packeting, when present at high concentrations, accurate quantitation of proteins can be affected due to detector saturation effects. Human plasma was analyzed in triplicate using liquid-chromatography data independent acquisition mass spectrometry (LC-DIA-MS) and using liquid-chromatography ion-mobility data independent acquisition mass spectrometry (LC-IM-DIA-MS). The inclusion of TWIMS was assessed for the effect on sample throughput, data integrity, confidence of protein and peptide identification, and dynamic range. The number of identified proteins is significantly increased by an average of 84% while both the precursor and product mass accuracies are maintained between the modalities. Sample dynamic range is also maintained while quantitation is achieved for all but the most abundant proteins by incorporating a novel data interpretation method that allows accurate quantitation to occur. This additional separation is all achieved within a workflow with no discernible deleterious effect on throughput. Consequently, TWIMS greatly enhances proteome coverage and can be reliably used for quantification when using an alternative product ion quantification strategy. Using TWIMS in biomarker discovery in human plasma is thus recommended.

A “plug-and-use” approach towards facile fabrication of capillary columns for high performance nanoflow liquid chromatography

Capillary columns used for nanoflow liquid chromatography play an important role in modern proteomics. High quality columns are needed to provide high peak capacity and highly reproducible separations. This is extremely important when multiple separations were compared in parallel in searching for potential biomarkers. Herein, we introduce a “plug-and-use” fritting technology for fabrication of high quality and highly reproducible capillary columns. Due to the identical length, good permeability, and stability of the prefabricated frits adopted, the capillary columns presented excellent performance consistency in terms of retention time, peak width as well as peak capacity at a column-to-column level (relative standard deviations, RSDs, at 0.4–0.9%, 2.1–3.6%, and 2.7%, respectively, n=6) for separations of complex mixtures of protein digest. For capillary columns packed with 5μm particles, high separation efficiency was demonstrated by the minimum plate height of 11μm, approaching the theoretical performance limit of such material. For separations of protein digests, the columns demonstrated excellent peak capacities of 110 and 300 for 20 and 360min gradients, respectively. The simple fabrication, good performance as well as consistent quality of such columns provide a reliable tool for high throughput separations requiring the use of multiple high performance capillary columns in parallel.

Proteomics Reveals Age-Related Differences in the Host Immune Response to Sepsis

Sepsis is commonly caused by community-acquired pneumonia (CAP) and may develop into severe sepsis, characterized by multiple organ failure. The risk of severe sepsis among CAP patients and subsequent mortality increases sharply after the age of 65. The molecular mechanisms associated with this age-related risk are not fully understood. To better understand factors involved with increased incidence and mortality of severe sepsis in the elderly, we used a nested case-control study of patients enrolled in a multicenter observational cohort of 2320 participants with CAP. We identified a total of 39 CAP patients 50-65 and 70-85 years old who did or did not develop severe sepsis. Plasma samples were obtained on presentation to the emergency department and prior to therapeutic interventions. A semiquantitative plasma proteomics workflow was applied which incorporated tandem immunoaffinity depletion, iTRAQ labeling, strong cation exchange fractionation, and nanoflow liquid chromatography coupled to high-resolution mass spectrometry. In total, 772 proteins were identified, of which 58 proteins exhibit statistically significant differences in expression levels among patients with severe sepsis as a function of age. Differentially expressed proteins are involved in pathways such as acute phase response, coagulation signaling, atherosclerosis signaling, lipid metabolism, and production of nitric oxide and reactive oxygen species. This study provides insight into factors that may explain age-related differences in incidence of severe sepsis in the elderly.

Noninvasive measurement of smoking-associated N3-ethyladenine and N7-ethylguanine in human salivary DNA by stable isotope dilution nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry

Evidence showed that ethylating agents are contained in cigarette smoke, which damage DNA producing ethylated DNA adducts, including N3-ethyladenine (3-EtAde) and N7-ethylguanine (7-EtGua). These two ethylpurines can be depurinated spontaneously and be repaired by enzymes and they have been detected in human urine. In this study, a highly specific and sensitive assay based on stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC–NSI/MS/MS) was used to measure 3-EtAde and 7-EtGua in human salivary DNA. These ethylpurines were released from DNA by neutral thermal hydrolysis and then enriched by a solid-phase extraction column before nanoLC–NSI/MS/MS analysis. The detection limits (S/N≥3) of 3-EtA and 7-EtG were 15fg (92amol) and 10fg (56amol), respectively, injected on-column. The lower quantification limits of 3-EtAde and 7-EtGua were both 100fg, i.e. 620 and 560amol, respectively, corresponding to 9.4 and 8.6 adducts in 109 normal nucleotides, respectively, starting with as little as 20μg of DNA isolated from an average of 3mL of saliva. The mean (±SD) levels of 3-EtAde in 15 smokers and 15 nonsmokers were 12.6±7.0 and 9.7±5.3 in 108 normal nucleotides, respectively, while those of 7-EtGua were 14.1±8.2 and 3.8±2.8 in 108 normal nucleotides in smokers and nonsmokers, respectively. Levels of 7-EtGua, but not 3-EtAde, were statistically significantly higher in smokers than in nonsmokers (p<0.0001). Furthermore, salivary 7-EtGua levels are significantly correlated with the number of cigarettes smoked per day as well as with the smoking index. This highly specific and sensitive stable isotope dilution nanoLC–NSI/MS/MS assay might be feasible in measuring 7-EtGua in human salivary DNA as a noninvasive biomarker for DNA damage induced by cigarette smoking.

Simultaneous quantification of ethylpurine adducts in human urine by stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry

Ethylating agents contained in cigarette smoke can damage DNA producing ethylated DNA adducts, including N3-ethyladenine (3-EtAde) and N7-ethylguanine (7-EtGua). In this study, a highly specific and sensitive assay based on stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) was used to measure 3-EtAde and 7-EtGua in human urine. These urinary adducts were enriched by a polymeric reversed phase solid-phase extraction column before the nanoLC-NSI/MS/MS analysis. The on-column detection limits (S/N≥3) of 3-EtAde and 7-EtGua were 15fg (92amol) and 10fg (56amol), respectively, while the lower quantification limits of 3-EtAde and 7-EtGua were 930 and 840 amol, respectively. Urinary concentrations of 3-EtAde and 7-EtGua in 21 smokers were 68.6±29.4 and 18.7±13.8pg/mL, respectively. In 20 nonsmokers, concentrations of 3-EtAde and 7-EtGua were 3.5±3.8 and 2.4±3.0pg/mL, respectively. The urinary concentrations of 3-EtAde and 7-EtGua were statistically significantly higher in smokers than in nonsmokers (p<0.0001). Moreover, 3-EtAde and 7-EtGua concentrations are significantly correlated with the number of cigarettes smoked per day and with the smoking index. This highly specific and sensitive assay based on stable isotope dilution nanoLC-NSI/MS/MS assay should be clinically valuable in assessing the possibility of measuring urinary ethylpurines as noninvasive biomarkers for smoking-related cancers in humans.

Detection and simultaneous quantification of three smoking-related ethylthymidine adducts in human salivary DNA by liquid chromatography tandem mass spectrometry

Smoking cigarette increases levels of certain ethylated DNA adducts in certain tissues and urine. Cigarette smoking is a major risk factor of various cancers and DNA ethylation is involved in smoking-related carcinogenesis. Among the ethylated DNA adducts, O2-ethylthymidine (O2-edT) and the promutagenic O4-ethylthymidine (O4-edT) are poorly repaired and they can accumulate in vivo. Using an accurate, highly sensitive, and quantitative assay based on stable isotope dilution nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry (nanoLC–NSI/MS/MS), O2-edT, N3-edT (N3-ethylthymidine), and O4-edT adducts in human salivary DNA were simultaneous detected and quantified. Saliva is easily accessible and available and it can be a potential target in searching for noninvasive biomarkers. Under the highly selected reaction monitoring (H-SRM) mode, salivary samples from 20 smokers and 13 nonsmokers were analyzed. Starting with 50μg of DNA isolated from about 3.5mL of saliva, levels of O2-edT, N3-edT, and O4-edT in 20 smokers’ salivary DNA samples were 5.3±6.2, 4.5±5.7, 4.2±8.0 in 108 normal nucleotides, respectively, while those in 13 nonsmokers were non-detectable. In addition, statistically significant correlations (p<0.0001) were observed between levels of O2-edT and N3-edT (γ=0.7388), between levels of O2-edT and O4-edT (γ=0.8839), and between levels of N3-edT, and O4-edT (γ=0.7835). To the best of our knowledge, this is the first report of detection and quantification of these three ethylthymidine adducts in human salivary DNA, which might be potential biomarkers for exposure to ethylating agents and possibly for cancer risk assessment.

Metaproteome Analysis of Endodontic Infections in Association with Different Clinical Conditions

Analysis of the metaproteome of microbial communities is important to provide an insight of community physiology and pathogenicity. This study evaluated the metaproteome of endodontic infections associated with acute apical abscesses and asymptomatic apical periodontitis lesions. Proteins persisting or expressed after root canal treatment were also evaluated. Finally, human proteins associated with these infections were identified. Samples were taken from root canals of teeth with asymptomatic apical periodontitis before and after chemomechanical treatment using either NaOCl or chlorhexidine as the irrigant. Samples from abscesses were taken by aspiration of the purulent exudate. Clinical samples were processed for analysis of the exoproteome by using two complementary mass spectrometry platforms: nanoflow liquid chromatography coupled with linear ion trap quadrupole Velos Orbitrap and liquid chromatography-quadrupole time-of-flight. A total of 308 proteins of microbial origin were identified. The number of proteins in abscesses was higher than in asymptomatic cases. In canals irrigated with chlorhexidine, the number of identified proteins decreased substantially, while in the NaOCl group the number of proteins increased. The large majority of microbial proteins found in endodontic samples were related to metabolic and housekeeping processes, including protein synthesis, energy metabolism and DNA processes. Moreover, several other proteins related to pathogenicity and resistance/survival were found, including proteins involved with adhesion, biofilm formation and antibiotic resistance, stress proteins, exotoxins, invasins, proteases and endopeptidases (mostly in abscesses), and an archaeal protein linked to methane production. The majority of human proteins detected were related to cellular processes and metabolism, as well as immune defense. Interrogation of the metaproteome of endodontic microbial communities provides information on the physiology and pathogenicity of the community at the time of sampling. There is a growing need for expanded and more curated protein databases that permit more accurate identifications of proteins in metaproteomic studies.

Separation of intact proteins by using polyhedral oligomeric silsesquioxane based hybrid monolithic capillary columns

High-efficient separation of intact proteins is still a huge challenge in proteome analysis of complex biological samples by using capillary columns. In this study, four POSS-based hybrid monolithic capillary columns were prepared and applied in nano-flow liquid chromatography (Nano-LC) separation of intact proteins. It was observed that the POSS-based hybrid monolithic columns exhibit high permeability, good LC separation reproducibility and column efficiency for intact protein separation. The effects of different LC separation conditions such as flow rate, gradient steepness, column length and mobile phase additives on the LC separation efficiency of the POSS-based hybrid monolithic column were systematically examined. Finally, fast LC separation of 7 proteins mixture was realized in 2.5min by using the optimized conditions on the 100μm i.d. POSS-based hybrid monolithic capillary column.

Significant modulation of the hepatic proteome induced by exposure to low temperature inXenopus laevis

SummaryThe African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control) and a low environmental temperature (5°C, cold exposure). Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism.

A SILAC-based Approach Identifies Substrates of Caspase-dependent Cleavage upon TRAIL-induced Apoptosis

The extracellular ligand-induced extrinsic pathway of apoptosis is executed via caspase protease cascades that activate downstream effectors by means of site-directed proteolysis. Here we identify proteome changes upon the induction of apoptosis by the cytokine tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a Jurkat T cell line. We detected caspase-dependent cleavage substrates by quantifying protein intensities before and after TRAIL induction in SDS gel slices. Apoptotic protein cleavage events are identified by a characteristic stable isotope labeling with amino acids in cell culture (SILAC) ratio pattern across gel slices that results from differential migration of the cleaved and uncleaved proteins. We applied a statistical test to define apoptotic substrates in the proteome. Our approach identified more than 650 of these cleaved proteins in response to TRAIL-induced apoptosis, including many previously unknown substrates and cleavage sites. Inhibitor treatment combined with triple SILAC demonstrated that the detected cleavage events were caspase dependent. Proteins located in the lumina of organelles such as mitochondria and endoplasmic reticulum were significantly underrepresented in the substrate population. Interestingly, caspase cleavage is generally observed in not only one but several members of stable complexes, but often with lower stoichiometry. For instance, all five proteins of the condensin I complex were cleaved upon TRAIL treatment. The apoptotic substrate proteome data can be accessed and visualized in the MaxQB database and might prove useful for basic and clinical research into TRAIL-induced apoptosis. The technology described here is extensible to a wide range of other proteolytic cleavage events.

Comparison of milk oligosaccharides between goats with and without the genetic ability to synthesize αs1-casein

Milk oligosaccharides (OS)—free complex carbohydrates—confer unique health benefits to the nursing neonate. Though human digestive enzymes cannot degrade these sugars, they provide nourishment to specific commensal microbes and act as decoys to prevent the adhesion of pathogenic micro-organisms to gastrointestinal cells. At present, the limited quantities of human milk oligosaccharides (HMO) impede research on these molecules and their potential applications in functional food formulations. Considerable progress has been made in the study of OS structures; however, the synthetic pathways leading to their synthesis in the mammary gland are poorly understood. Recent studies show that complex OS with fucose and N-acetyl neuraminic acid (key structural elements of HMO bioactivity) exist in goat milk. Polymorphisms in the CSN1S1 locus, which is responsible for synthesis of αs1-casein, affect lipid and casein micelle structure in goat milk. The present study sought to determine whether CSN1S1 polymorphisms also influence goat milk oligosaccharide (GMO) production and secretion. The GMO compositions of thirty-two goat milk samples, half of which were from genotype A/A (αs1-casein producers) and half from genotype O/O (αs1-casein non-producers), were determined with nanoflow liquid chromatography high-accuracy mass spectrometry. This study represents the most exhaustive characterization of GMO to date. A systematic and comprehensive GMO library was created, consolidating information available in the literature with the new findings. Nearly 30 GMO, 11 of which were novel, were confirmed via tandem mass spectrometric analyses. Six fucosylated OS were identified; 4 of these matched HMO compositions and three were identified for the first time in goat milk. Importantly, multivariate statistical analysis demonstrated that the OS profiles of the A/A and O/O genotype milks could be discriminated by the fucosylated OS. Quantitative analysis revealed that the goat milk samples contained 1.17g/L of OS; however, their concentration in milks from A/A and O/O genotypes was not different. This study provides evidence of a genetic influence on specific OS biosynthesis but not total OS production. The presence of fucosylated GMO suggests that goat milk represents a potential source of bioactive milk OS suitable as a functional food ingredient.

Proteomic analysis of differentially expressed proteins in caprine milk during experimentally induced endotoxin mastitis

The goal of the current study was to identify proteins in goat milk before and at 18h following intramammary challenge with lipopolysaccharide (LPS). Initial evaluation of protein profiles generated using 2-dimensional gel electrophoresis on skim milk samples from a group of 6 goats collected before challenge and at 18, 24, and 48h after LPS challenge revealed little change in the abundance of casein proteins, and minimal changes in the presence or abundance of the plasma protein serum albumin, which is known to leak into milk during coliform mastitis in dairy cattle. Proteins in baseline milk samples and in milk from the same goats 18h post-LPS challenge were excised from the gels, and peptides were sequenced using nano-flow liquid chromatography coupled with tandem mass spectrometry. Despite the overwhelming presence of casein proteins and β-lactoglobulin, the lower abundance proteins β-2-microglobulin, fatty acid-binding protein, serum albumin, and retinol-binding protein were detected in skim milk samples from healthy goats. Skim milk samples 18h postchallenge were characterized by the sustained presence and abundance of the casein proteins, and by the presence of haptoglobin, serum amyloid A, lactoferrin, cathelicidin-1, and cathelicidin-3. No marked differences in the intensity of the spot corresponding to serum albumin were observed in gels of skim milk samples 18h postchallenge, which could indicate that the breakdown of the blood–milk barrier during endotoxin mastitis may not be as profound in goats as has been observed in dairy cattle. Nonetheless, the occurrence of an inflammatory response was supported by elevated somatic cell counts in the goat milk following inoculation with endotoxin, as well as by the presence of both antimicrobial and acute phase proteins. The results provide information about the composition of proteins in goat milk as well as added knowledge of the host response during endotoxin mastitis in goats.

Characterization of oxidized phospholipids in oxidatively modified low density lipoproteins by nanoflow liquid chromatography–tandem mass spectrometry

Oxidized low density lipoproteins (Ox-LDLs) have an important role in the development of age-related vascular disease, such as atherosclerosis. Ox-LDLs are defined as oxidatively modified LDLs in the blood by enzymatic or non-enzymatic oxidation of phospholipids (PLs). For the characterization of Ox-LDLs at molecular level, oxidation patterns of oxidized PL (Ox-PL) products were systematically examined with standard PL molecules (16:0/22:6-PC, 18:0/22:6-PA, and 18:0/22:6-PG), by the formation of bilayer vesicles of each standard, followed by oxidation of PL vesicles using a Cu2+ solution. This oxidative modification was applied to LDL standard materials. Nanoflow liquid chromatography–electrospray ionization–tandem mass spectrometry (nLC–ESI–MS/MS) analysis of the extracted PL mixtures resulted in the identification of 276 PLs both in the modified and non-modified LDL, including 139 Ox-PL species. Examination of the identified PL species from the standard LDL before and after oxidation supported the postulate that the specific location of an acyl chain of LPL can be exchanged between the sn-1 and -2 positions. This exchange occurs when the neighboring acyl chain is cleaved during oxidation and the polar head group of PL molecules can be dissociated to form PA molecules that result in the formation of various Ox-PA products. This study demonstrates that nLC–ESI–MSn can be utilized for the separation and structural characterization of complicated Ox-PL mixtures, including long chain products with hydroxylation or hydroperoxylation at an unsaturated acyl chain, and short chain products from the cleavage of unsaturated fatty acyl chains to form lysophospholipids (LPLs) or the truncation of an acyl chain into a shorter chain terminated with aldehyde or carboxylic acid.

Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.

A quantitative and comprehensive method to analyze human milk oligosaccharide structures in the urine and feces of infants

Human milk oligosaccharides (HMOs), though non-nutritive to the infant, shape the intestinal microbiota and protect against pathogens during early growth and development. Infant formulas with added galacto-oligosaccharides have been developed to mimic the beneficial effects of HMOs. Premature infants have an immature immune system and a leaky gut and are thus highly susceptible to opportunistic infections. A method employing nanoflow liquid chromatography time-of-flight mass spectrometry (MS) is presented to simultaneously identify and quantify HMOs in the feces and urine of infants, of which 75 HMOs have previously been fully structurally elucidated. Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS was employed for high-resolution and rapid compositional profiling. To demonstrate this novel method, samples from mother-infant dyads as well as samples from infants receiving infant formula fortified with dietary galacto-oligosaccharides or probiotic bifidobacteria were analyzed. Ingested oligosaccharides are demonstrated in high abundance in the infant feces and urine. While the method was developed to examine specimens from preterm infants, it is of general utility and can be used to monitor oligosaccharide consumption and utilization in term infants, children, and adults. This method may therefore provide diagnostic and therapeutic opportunities.