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The glycoprotein uromodulin (UMOD) is the most abundant protein in urine, and N-glycans are critical for many biological functions of UMOD. Comprehensive glycan profiling of UMOD provides valuable information to understand the exact mechanisms of glycan-regulated functions. To perform comprehensive glycosylation analysis of UMOD from urine samples with limited volumes, we developed a streamlined workflow that included UMOD isolation from 5 mL of urine from 6 healthy adult donors (3 males and 3 females) and a glycosylation analysis using a highly sensitive and reproducible nanoLC-MS/MS based glycomics approach. In total, 212 N-glycan compositions were identified from the purified UMOD, and 17% were high-mannose glycans, 2% were afucosylated/asialylated, 3% were neutral fucosylated, 28% were sialylated (with no fucose), 46% were fucosylated and sialylated, and 4% were sulfated. We found that isolation of UMOD resulted in a significant decrease in the relative quantity of high-mannose and sulfated glycans with a significant increase of neutral fucosylated glycans in the UMOD-depleted urine relative to the undepleted urine, but depletion had little impact on the sialylated glycans. To our knowledge, this is the first study to perform comprehensive N-glycan profiling of UMOD using nanoLC-MS/MS. This analytical workflow would be very beneficial for studies with limited sample size, such as pediatric studies, and can be applied to larger patient cohorts not only for UMOD interrogation but also for global glycan analysis.

Recurrent urinary tract infections (UTIs) pose a significant burden on the health care system. Underlying mechanisms predisposing children to UTIs and associated changes in the urinary proteome are not well understood. We aimed to investigate the urinary proteome of a subset of children who have vesicoureteral reflux (VUR) and recurrent UTIs because of their risk of developing infection-related renal damage. Improving diagnostic modalities to identify UTI risk factors would significantly alter the clinical management of children with VUR. We profiled the urinary proteomes of 22 VUR patients with low grade VUR (1–3 out of 5), a history of recurrent UTIs, and renal scarring, comparing them to those obtained from 22 age-matched controls. Urinary proteins were analyzed by mass spectrometry followed by protein quantitation based on spectral counting. Of the 2,551 proteins identified across both cohorts, 964 were robustly quantified, as defined by meeting criteria with spectral count (SC) ≥2 in at least 7 patients in either VUR or control cohort. Eighty proteins had differential expression between the two cohorts, with 44 proteins significantly up-regulated and 36 downregulated (q

A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories., Graphical Abstract Highlights A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb. 103 reports were received from 76 diverse laboratories worldwide. Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method. Consensus values were derived and similar performance across all experimental methods was noted., Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

The glycoprotein uromodulin (UMOD) is the most abundant protein in urine, and

Background and ObjectivesSport-related concussions affect millions of individuals across the United States each year, and current techniques to diagnose and monitor them rely largely on subjective measures. Our goal was to discover and validate objective, quantifiable noninvasive biomarkers with the potential to be used in sport-related concussion diagnosis.MethodsUrine samples from a convenience series of healthy control collegiate athletes who had not sustained a concussion and athletes who sustained a concussion as diagnosed by a sports medicine physician within 7 days were collected prospectively and studied. Participants also completed an instrumented single-task gait analysis as a functional measure. Participants were recruited from a single collegiate athletic program and were ≥18 years of age and were excluded if they had a concomitant injury, active psychiatric conditions, or preexisting neurologic disorders. Using Tandem Mass Tags (TMT) mass spectroscopy and ELISA, we identified and validated urinary biomarkers of concussion.ResultsForty-eight control and 47 age- and sex-matched athletes with concussion were included in the study (51.6% female, 48.4% male, average age 19.6 years). Participants represented both contact and noncontact sports. All but 1 of the postconcussion participants reported experiencing symptoms at the time of data collection. Insulin-like growth factor 1 (IGF-1) and IGF binding protein 5 (IGFBP5) were downregulated in the urine of athletes with concussions compared to healthy controls. Multivariable risk algorithms developed to predict the probability of sport-related concussion showed that IGF-1 multiplexed with single-task gait velocity predicts concussion risk across a range of postinjury time points (area under the curve [AUC] 0.786, 95% confidence interval [CI] 0.690–0.884). When IGF-1 and IGFBP5 are multiplexed with single-task gait velocity, they accurately distinguish between healthy controls and individuals with concussion at acute time points (AUC 0.835, 95% CI 0.701–0.968, p < 0.001).DiscussionThese noninvasive biomarkers, discovered in an objective and validated manner, may be useful in diagnosing and monitoring sport-related concussions in both acute phases of injury and several days after injury.Trial Registration InformationClinicalTrials.gov Identifier: NCT02354469 (submitted February 2015, first patient enrolled August 2015).Classification of EvidenceThis study provides Class III evidence that urinary IGF-1 and IGFBP5 multiplexed with single-task gait velocity may be useful in diagnosing sport-related concussion.

We performed an in-depth characterization and comparison of the pediatric and adult urinary glycomes using a nanoLC-MS/MS based glycomics method, which included normal healthy pediatric (1-10 years, n = 21) and adult (21-50 years, n = 22) individuals. A total of 116 N-glycan compositions were identified, and 46 of them could be reproducibly quantified. We performed quantitative comparisons of the 46 glycan compositions between different age and sex groups. The results showed significant quantitative changes between the pediatric and adult cohorts. The pediatric urinary N-glycome was found to contain a higher level of high-mannose (HM), asialylated/afucosylated glycans (excluding HM), neutral fucosylated and agalactosylated glycans, and a lower level of trisialylated glycans compared with the adult. We further analyzed gender-associated glycan changes in the pediatric and adult group, respectively. In the pediatric group, there was almost no difference of glycan levels between males and females. In adult, the majority of glycans were more abundant in males than females, except the high-mannose and tetrasialylated glycans. These findings highlight the importance to consider age-matching and adult sex-matching for urinary glycan studies. The identified normal pediatric and adult urinary glycomes can serve as a baseline reference for comparisons to other disease states affected by glycosylation.

Prenatal hydronephrosis is a common condition that may spontaneously resolve after birth. However, this condition can result in renal damage and requires surgical correction in a number of cases. Preventing renal damage is paramount, but existing diagnostic technology is invasive, exposes infants to radiation, is costly, and is often indeterminate. A better understanding of the pathophysiology of renal obstruction as reflected in the urinary proteome may provide new insights into the disease that could potentially alter the clinical management of hydronephrosis. We performed a quantitative proteomics study of urine that was surgically obtained from eight clinically significant, unilaterally obstructed infants versus eight healthy controls, with the goal of identifying quantitatively varying proteins and the biological networks associated with them. Notably, urine was obtained from both the obstructed kidney and the bladder. Over 1100 proteins were identified, and a total of 76 quantitatively varying proteins were identified. Proteins involved in oxidative stress, inflammation, and renal disease pathways showed the most significant abundance differences. This study gives a deeper understanding of the critical proteomic changes associated with renal obstruction and represents the deepest proteomic profile of renal obstruction to date.

The idiopathic nature of Urological Chronic Pelvic Pain Syndrome (UCPPS), which encompasses interstitial cystitis/bladder pain syndrome (IC/BPS) and chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), has prompted an intense search for clinical biomarkers. Identification of clinically relevant, validated biologic markers of UCPPS has great potential to inform our understanding of UCPPS pathophysiology, development, and progression, and ultimately clinical management. To date, the cause of this syndrome is unknown and there is no ability to determine who is at risk of progression or who will respond to specific therapy. Accurate noninvasive tests, based on objective, specific, and definable levels of validated biomarkers, are essential to improve and standardized clinical guidelines and for more targeted management strategies based on patient profiles. The MAPP Research Network was established by the NIDDK of the NIH and represents a comprehensive approach to the study of UCPPS 1, 2. A major goal of this initiative is to better understand pathophysiology and identify potential therapeutic targets. In this MAPP Network study, we have utilized a biologically-driven, candidate biomarker discovery strategy, grounded in the basic biochemistry and physiology of this syndrome, to evaluate candidate non-invasive biomarkers of UCPPS. Reviews of the literature have revealed a number of candidate protein biomarker targets. Increased VEGF levels have been detected in bladder biopsy samples from patients with IC compared to controls, with levels correlating with pain severity 3, 4. Prior studies also suggest an important connection between neovascularization and IC, in that new blood vessels in biopsy samples of IC patients showed significantly lower levels of pericyte coverage of the nascent endothelium, evidence consistent with the known association between high levels of VEGF and immature vessel formation 3. Moreover, VEGF originally discovered as VPF (Vascular Permeability Factor), 5, 6 is the most potent endogenous vascular permeability factor and may mediate the dysregulated vascular permeability postulated to be important in UCPPS 7–9. Given these associations, we chose to include VEGF and its receptor VEGFR1, as biomarker candidates. Inflammation has been suggested to be an underlying pathophysiologic mechanism in UCPPS 10 and increased concentrations of pro-inflammatory cytokines (IL-1β, TNF-α, IL-8) have been reported in patients with UCPPS as compared with controls 11, 12. In this respect, MMPs are of particular interest. Originally thought to be associated exclusively with tissue remodeling and destruction, it is now widely appreciated that MMPs play a role in a variety of biologic processes including cytokine and growth factor release, tumor growth and progression, angiogenesis and a number of inflammatory conditions 13–17. During the inflammatory response, MMPs and their complexes are released from connective tissue cells in response to pro-inflammatory cytokines. Upregulation of MMP activity results in the recruitment of pro-inflammatory cells to the site of tissue injury. MMPs have also been implicated in the regulation of the immune response because of their ability to cleave inflammatory mediators and stimulate the clearance of inflammatory cells 18–21. Given that MMPs may play an important role in UCPPS as a function of the inflammatory phenotype, abnormal vasculature and the dysregulation of ECM (extracellular matrix) turnover present in this and other potentially related diseases we included these proteins in our study. NGAL, also known as Lipocalin-2 (Lcn2), is upregulated in numerous chronic and acute inflammatory conditions, including chronic kidney disease, ulcerative colitis and myocardial infarction 22, 23. There exists an intricate interplay between NGAL and other inflammatory cytokines as well, such that NGAL upregulation can be triggered by multiple inflammatory cytokines, including TNF-α, IL-1β and IL-17 22, 23. Moreover, NGAL itself is a modulator of the levels of other inflammatory cytokines, as well as of the behavior of inflammatory leukocytes 24–26. In our laboratory, we have demonstrated that NGAL is a significant stimulator of VEGF levels in breast tumor cells and can also independently induce the epithelial to mesenchymal transition in breast cancer, as well as being a noninvasive biomarker of this disease 27–29. NGAL can also form a complex with MMP-9, representing a distinct protein complex referred to as MMP-9/NGAL complex. We have previously shown that when NGAL complexes with MMP-9, it protects MMP-9 from degradation preserving its enzyme activity 16, 30. The MMP-9/NGAL complex has also been detected in urine samples from patients with various diseases. Based on these findings, we chose to measure NGAL as well as MMP-9/NGAL complex in the urine of individuals with UCPPS and correlate them with clinical symptoms. Here, we report sex-specific comparisons of candidate biomarkers among UCPPS and control cohorts to gain new insights into underlying pathophysiology. In addition, associations of biomarkers with symptom severity within the UCPPS cohort are examined. We anticipate that the identified biological markers may serve as potential candidates for further clinical evaluation, including in the development of new treatment strategies directed toward novel targets, as objective measures for patient classification schemes, and evaluation of clinical outcomes, as well as further characterization of underlying mechanism.

Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-(13)[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans.

The past 15 years have seen significant progress in LC-MS/MS peptide sequencing, including the advent of successful de novo and database search methods; however, analysis of glycopeptide and, more generally, glycoconjugate spectra remains a much more open problem, and much annotation is still performed manually. This is partly because glycans, unlike peptides, need not be linear chains and are instead described by trees. In this study, we introduce SweetSEQer, an extremely simple open source tool for identifying potential glycopeptide MS/MS spectra. We evaluate SweetSEQer on manually curated glycoconjugate spectra and on negative controls, and we demonstrate high quality filtering that can be easily improved for specific applications. We also demonstrate a high overlap between peaks annotated by experts and peaks annotated by SweetSEQer, as well as demonstrate inferred glycan graphs consistent with canonical glycan tree motifs. This study presents a novel tool for annotating spectra and producing glycan graphs from LC-MS/MS spectra. The tool is evaluated and shown to perform similarly to an expert on manually curated data.

We examined symptom variability in men and women with urological chronic pelvic pain syndrome. We describe symptom fluctuations as related to early symptom regression and its effect on estimated 1-year symptom change. We also describe a method to quantify patient specific symptom variability.Symptoms were assessed biweekly in 424 subjects with urological chronic pelvic pain syndrome during 1 year. To evaluate the impact of early symptom regression subjects were classified as improved, no change or worse according to the rate of change using 1) all data, 2) excluding week 0 and 3) excluding weeks 0 and 2. Patient specific, time varying variability was calculated at each interval using a sliding window approach. Patients were classified as high, medium or low variability at each time and ultimately as high or low variability overall based on the variability for the majority of contacts.Prior to excluding early weeks to adjust for early symptom regression 25% to 38% and 5% to 6% of patients were classified as improved and worse, respectively. After adjustment the percent of patients who were improved or worse ranged from 15% to 25% and 6% to 9%, respectively. High and low variability phenotypes were each identified in 25% to 30% of participants.Patients with urological chronic pelvic pain syndrome show symptom variability. At study enrollment patients had worse symptoms on average, resulting in a regression effect that influenced the estimated proportion of those who were improved or worse. Prospective studies should include a run-in period to account for regression to the mean and other causes of early symptom regression. Further, symptom variability may be quantified and used to characterize longitudinal symptom profiles of urological chronic pelvic pain syndrome.

INTRODUCTION AND OBJECTIVES: Stress urinary incontinence, a major type of urinary incontinence, increases with age and is often developed after partum injury. Low intensity pulsed ultrasound (LIPUS) has been investigated in the treatment of many diseases showing its ability of restoring soft tissue injury. We investigated the therapeutic effect of low intensity pulsed ultrasound in stress urinary incontinence. METHODS: Thirty-two Sprague Dawley rats in SUI group underwent vaginal distension (VD) and bilateral ovariectomy mimicking partum injury. Eight rats served as mock operation control. Eight rats each in SUI group was treated with low-dosage LESW (0.03 mJ/mm2), medium-dosage LESW (0.06 mJ/mm2), or high-dosage LESW (0.09 mJ/mm2). The rest eight rats served as none-treatment group. For functional study, leak point pressure test (LPP) was performed 2 weeks after the last LESW. Masson trichrome staining was performed to validate the pathological changes. RESULTS: The LPP was restored in medium-dosage LESW and high-dosage LESW groups, but not in low-dosage LESW group. More robust striated muscle regeneration was found in these two group comparing with the none-treatment group. CONCLUSIONS: LIPUS ameliorate the symptom of SUI via activating striated muscle regeneration.

Given the biological importance of glycosylation on proteins, the identification of protein glycosylation sites is integral to understanding broader biological structure and function. Unfortunately, the determination of the microheterogeneity at the site of glycosylation still remains a significant challenge. Nanoflow liquid chromatography with tandem mass spectrometry provides both separation of glycopep- tides and the ability to determine glycan composition and site-specific glycosylation. However, because of the size of glycopeptides, they are not often amenable to tandem MS. In this work, proteins are digested with multiple proteases to produce glycopeptides that are of suitable size for tandem MS analysis. The conditions for collision-induced dissociation are optimized to obtain diagnostic ions that maximize glycan and peptide information. The method is applied to glycoproteins with contrasting glycans and multiple sites of glycosylation and identifies multiple glycan compositions at each individual glycosylation site. This method provides an important improvement in the routine determination of glycan microheterogeneity by mass spectrometry.

Protein phosphorylation is a critical posttranslational modification that affects cell-cell signaling and protein function. However, quantifying the relative site-specific changes of phosphorylation occupancies remains a major issue. An online enrichment of phosphopeptides using titanium dioxide incorporated in a microchip liquid chromatography device was used to analyze trypsin-digested human milk proteins with mass spectrometry. The method was validated with standards and used to determine the dynamic behavior of protein phosphorylation in human milk from the first month of lactation. α-Casein, β-casein, osteopontin, and chordin-like protein 2 phosphoproteins were shown to vary during this lactation time in an independent manner. In addition, changes in specific regions of these phosphoproteins were found to vary independently. Novel phosphorylation sites were discovered for chordin-like protein 2, α-lactalbumin, β-1,4-galactosyl transferase, and poly-Ig (immunoglobulin) receptor. Coefficients of variation for the quantitation were comparable to those in other contemporary approaches using isotopically labeled peptides, with a median value of 11% for all phosphopeptide occupancies quantified.

Introduction Perfluorooctanesulfonate (PFOS) is widely distributed and persistent in humans and wildlife. Prior toxicological studies have reported decreased total and free thyroid hormones in serum without a major compensatory rise in thyrotropin (TSH) or altered thyroid gland histology. Although these animals (rats, mice and monkeys) might have maintained an euthyroid state, the basis for hypothyroxinemia remained unclear. We undertook this study to investigate the causes for the PFOS-induced reduction of serum total thyroxine (TT4) in rats. Hypotheses We hypothesized that exposure to PFOS may increase free thyroxine (FT4) in the rat serum due to the ability of PFOS to compete with thyroxine for binding proteins. The increase in FT4 would increase the availability of the thyroid hormone to peripheral tissues for utilization, metabolic conversation, and excretion. We also hypothesized that PFOS does not directly interfere with the regulatory functions of the hypothalamic–pituitary–thyroid (HPT) axis in rats. Experiments Three experimental designs were employed to test these hypotheses. (1) Female Sprague–Dawley (SD) rats were given a single oral dose of 15 mg potassium PFOS/kg body weight. At intervals of 2, 6, and 24 h thereafter, measurements were made for serum FT4, TT4, triiodothyronine (TT3), reverse triiodothyronine (rT3), thryrotropin (TSH), and PFOS concentrations, as well as liver PFOS concentrations, UDP-glucuronosyltransferase 1A (UGT1A) family mRNA transcripts, and malic enzyme (ME) mRNA transcripts and activity. (2) To provide evidence for increased uptake and metabolism of thyroxine (T4), 125I-T4 was given to male and female SD rats by intravenous injection, followed in 2 h by a single oral dose of 15 mg potassium PFOS/kg body weight. 125I radioactivity was determined in urine and feces collected over a 24-h period and in serum and liver collected at 24 h. (3) To assess the potentials effect of PFOS on the hypothalamic–pituitary–thyroid axis, over an 8-day period, groups of male SD rats were given PFOS (3 mg/kg-d), propyl thiouracil (PTU, 10 μg/mL in water), or PTU and PFOS in combination, with controls receiving 0.5% Tween® 20 vehicle. On days 1, 3, 7, and 8, TT4, TT3, and TSH were monitored. On day 8, pituitaries were removed and placed in static culture for assessment of thyrotropin releasing hormone (TRH)-mediated release of TSH. Results (1) PFOS transiently increased FT4 and decreased TSH within 6 h, with values returning to control levels by 24 h. TT4 was decreased by 55% over a 24-h period. TT3 and rT3 were decreased at 24 h to a lesser extent than TT4. ME mRNA transcripts were increased at 2 h and activity was increased at 24 h. UGT1A mRNA transcripts were increased at 2 and 6 h. (2) 125I decreased in serum and liver relative to controls and consistent with a reduction in serum TT4. Concomitantly, 125I activity was increased in urine and feces collected from PFOS-treated rats. (3) During the 8 days of dosing with PFOS, TSH was not elevated in male rats, while TT4 and TT3 were decreased. Pituitary response to TRH-mediated TSH release was not diminished after 8-daily oral doses of PFOS. Conclusions These findings suggest that oral dosing in rats with PFOS results in transiently increased tissue availability of the thyroid hormones and turnover of T4 with a resulting reduction in serum TT4. PFOS does not induce a classical hypothyroid state under dosing conditions employed nor does it alter HPT activities.

Synovial fluid in an articulating joint contains proteins derived from the blood plasma and proteins that are produced by cells within the joint tissues, such as synovium, cartilage, ligament, and meniscus. The proteome composition of healthy synovial fluid and the cellular origins of many synovial fluid components are not fully understood. Here, we present a normative proteomics study using porcine synovial fluid. Using our optimized method, we identified 267 proteins with high confidence in healthy synovial fluid. We also evaluated mRNA expression data from tissues that can contribute to the synovial fluid proteome, including synovium, cartilage, blood, and liver, to better estimate the relative contributions from these sources to specific synovial fluid components. We identified 113 proteins in healthy synovial fluid that appear to be primarily derived from plasma transudates, 37 proteins primarily derived from synovium, and 11 proteins primarily derived from cartilage. Finally, we compared the identified synovial fluid proteome to the proteome of human plasma, and we found that the two body fluids share many similarities, underlining the detected plasma derived nature of many synovial fluid components. Knowing the synovial fluid proteome of a healthy joint will help to identify mechanisms that cause joint disease and pathways involved in disease progression.

Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either N-glycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of N-glycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed de-N-glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including de-N-glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin, and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their nonsugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, glycoprotein quantification based exclusively on deglycosylated peptides may lead to inaccurate quantification.

In this study, we performed an in-depth characterization of the male pediatric infant urinary proteome by parallel proteomic analysis of normal healthy adult (n=6) and infant (n=6) males and comparison to available published data. A total of 1584 protein groups were identified. Of these, 708 proteins were identified in samples from both cohorts. Although present in both cohorts, 136 of these common proteins were significantly enriched in urine from adults and 94 proteins were significantly enriched in urine from infants. Using Gene Ontology, we found that the infant-enriched or specific subproteome (743 proteins) had an overrepresentation of proteins that are involved in translation and transcription, cellular growth and metabolic processes. In contrast, the adult enriched or specific subproteome (364 proteins) showed an overexpression of proteins involved in immune response and cell adhesion. This study demonstrates that the non-diseased male urinary proteome is quantitatively affected by age, has age-specific subproteomes, and identifies a common subproteome with no age-dependent abundance variations. These findings highlight the importance of age-matching in urinary proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Determining which glycan moieties occupy specific N-glycosylation sites is a highly challenging analytical task. Arguably, the most common approach involves LC-MS and LC-MS/MS analysis of glycopeptides generated by proteases with high cleavage site specificity; however, the depth achieved by this approach is modest. Nonglycosylated peptides are a major challenge to glycoproteomics, as they are preferentially selected for data-dependent MS/MS due to higher ionization efficiencies and higher stoichiometric levels in moderately complex samples. With the goal of improving glycopeptide coverage, a mass defect classifier was developed that discriminates between peptides and glycopeptides in complex mixtures based on accurate mass measurements of precursor peaks. By using the classifier, glycopeptides that were not fragmented in an initial data-dependent acquisition run may be targeted in a subsequent analysis without any prior knowledge of the glycan or protein species present in the mixture. Additionally, from probable glycopeptides that were poorly fragmented, tandem mass spectra may be reacquired using optimal glycopeptide settings. We demonstrate high sensitivity (0.892) and specificity (0.947) based on an in silico dataset spanning >100,000 tryptic entries. Comparable results were obtained using chymotryptic species. Further validation using published data and a fractionated tryptic digest of human urinary proteins was performed, yielding a sensitivity of 0.90 and a specificity of 0.93. Lists of glycopeptides may be generated from an initial proteomics experiment, and we show they may be efficiently targeted using the classifier. Considering the growing availability of high accuracy mass analyzers, this approach represents a simple and broadly applicable means of increasing the depth of MS/MS-based glycoproteomic analyses.

Common de-N-glycosylation protocols usually require a lengthy incubation time. Although pressure cycling technology or scientific microwave reactors can accelerate this enzyme reaction, they may not be easily accessible. In this brief report, we employed an alternative strategy using a standard domestic microwave oven to perform the de-N-glycosylation. Model glycoproteins (bovine RNase B, bovine fetuin, and human IgG) and a complex mixture from human plasma were fully deglycosylated in 20 min, without any apparent adverse affects on the glycans or protein backbones. This new method provides a simple and inexpensive solution to achieve rapid de-N-glycosylation.

Milk fat globules (MFGs) are accepted primarily as triacylglycerol delivery systems. The identification of nanometer-sized lipid-protein particles termed "lactosomes" that do not contain triacylglycerol raises the question of their possible functions. MFGs were isolated by slow centrifugation, and lactosomes were isolated by ultracentrifugation at a density equivalent to plasma high-density lipoproteins (HDL) (d > 1.063 g/mL) from human milk obtained from six volunteers at different lactation stages. Isolated lactosomes were analyzed and compared with MFGs for their size distribution, lipidome, proteome, and functional activity. Lactosomes from early milk, day 8, were found to be similar in size as those from mature milk >28 days, averaging ∼ 25 nm in diameter. In total, 97 nonredundant proteins were identified in the MFG and lactosome fractions, 46 of which were unique to the MFG fraction and 29 of which were unique to the lactosome fraction. The proteins identified in the lactosome and MFG fractions were enriched with proteins identified with immunomodulatory pathways. Unlike MFGs and GM1-laden reconstituted HDL that served as a positive control, lactosomal binding capacity to cholera toxin was weak. Lipidomic analyses found that lactosomes were devoid of triacylglycerol and gangliosides, unlike MFGs, but rich in a variety of phospholipid species. The data found differences in structure, composition, and function between lactosomes and MFG, suggesting that these two particles are derived from different biosynthetic and/or secretory pathways. The results reveal a bioactive lipid-protein, nanometer-length scale particle that is secreted into milk not to supply energy to the infant but to play unique, protective, and regulatory roles.

While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first ten days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor.

Human milk contains large amounts of complex oligosaccharides that putatively modulate the intestinal microbiota of breast-fed infants by acting as decoy binding sites for pathogens and as prebiotics for enrichment of beneficial bacteria. Several bifidobacterial species have been shown to grow well on human milk oligosaccharides. However, little data exists on other bacterial species. In this work we examined 16 bacterial strains belonging to 10 different genera for growth on human milk oligosaccharides. For this propose, we used a chemically-defined medium, ZMB1, which allows vigorous growth of a number gut–related microorganisms in a fashion similar to complex media. Interestingly, Bifidobacterium longum subsp. infantis, Bacteroides fragilis and Bacteroides vulgatus strains were able to metabolize milk oligosaccharides with high efficiency, while Enterococcus, Streptococcus, Veillonella, Eubacterium, Clostridium, and Escherichia coli strains grew less well or not at all. Mass spectrometry-based glycoprofiling of the oligosaccharide consumption behavior revealed a specific preference for fucosylated oligosaccharides by Bifidobacterium longum subsp. infantis and Bacteroides vulgatus. This work expands the current knowledge of human milk oligosaccharides consumption by gut microbes, revealing bacteroides as avid consumer of this substrate. These results provide insight on how human milk oligosaccharides shape the infant intestinal microbiota.

Perfluorooctanesulfonate [PFOS; CF3(CF2)7SO3−] and its acid salts were derived from perfluorooctanesulfonyl fluoride [POSF; CF3(CF2)7SO2F]. Major product applications were developed using POSF through formation of N-alkylsulfonamides that were used in surfactants, paper and packaging treatments, and surface protectants (e.g., carpet, upholstery, textiles). Depending on the specific functional derivitization or polymerization, these POSF-based products may have degraded or metabolized, to an undetermined degree, to PFOS, a stable and persistent end product that has a widespread presence in the general population (Butenhoff et al. 2006) and wildlife (Houde et al. 2006). Salts of perfluorooctanoic acid, in particular ammonium perfluorooctanoate (APFO), have been used as surfactants and processing aids in the production of fluoropolymers and fluoro-elastomers. Industrial production of the salts of perfluorooctanoic acid occur through electrochemical fluorination and telomerization. Perfluorooctanoate [PFOA; CF3(CF2)6COO−], the dissociated carboxylate anion, has been measured in humans worldwide but generally at lower nanogram per milliliter concentrations than PFOS (Houde et al. 2006). In rats, PFOS and PFOA are not metabolized and enter into the enterohepatic circulation (Johnson et al. 1984; Kemper 2003; Kuslikis et al. 1992; Vanden Heuvel et al. 1991). Because of the stability of the carbon–fluorine bond and the high electronegativity of perfluorinated alkyl acids, metabolism would not be favored; thus, perfluorohexanesulfonate (PFHS) is also not expected to be metabolized. Based on the determination of volumes of distribution from single-dose intravenous studies in cynomolgus monkeys, the distributions of PFOS, PFHS, and PFOA are primarily extracellular (Butenhoff et al. 2004; Noker and Gorman 2003a, 2003b). Kerstner-Wood et al. (2003) found PFOS, PFHS, and PFOA to be highly bound in rat, monkey, and human plasma over a concentration range of 1–500 μg/mL. When incubated with human plasma protein fractions, all three compounds were highly bound (99.7 to > 99.9%) to albumin, and showed affinity for β-lipoproteins (95.6, 64.1, and 39.6% for PFOS, PFHS, and PFOA, respectively). Some binding to α - and γ -globulin fractions and minor interactions with transferrin (PFHS and PFOA) were also noted. PFOS and PFOA have been shown to compete for fatty acid binding sites on liver fatty acid binding protein, with PFOS giving the stronger response (Luebker et al. 2002). The elimination rates of PFOS and PFHS have been studied in male and female cynomolgus monkeys after intravenous dosing (Noker and Gorman 2003a, 2003b) and for PFOS after repeated oral dosing (Seacat et al. 2002). Noker and Gorman (2003a, 2003b) reported mean (± SD) terminal elimination half-lives, ranging from 88 to 146 days (132 ± 13 days for males and 110 ± 26 days for females) for PFOS and 49 to 200 days (141 ± 52 days for males and 87 ± 47 days for females) for PFHS, after intravenous dosing of three male and three female cynomolgus monkeys in separate experiments, with no significant difference between males and females or between the two compounds. Seacat et al. (2002) reported an approximate terminal elimination half-life of 200 days for PFOS in male and female cynomolgus monkeys during 1 year immediately following 6 months of daily oral dosing with either 0.15 or 0.75 mg/kg PFOS. Elimination rates in species other than the monkey have been determined for PFOS and PFOA. Within 89 days after a single intravenous dose of 14C-PFOS, 30% of the 14C was excreted in the urine and 12% in the feces of male rats (Johnson et al. 1979). For PFOA, significant interspecies differences have been observed (Hundley et al. 2006; Kudo and Kawashima 2003), and differential expression of organic anion transporters in renal proximal tubule cells have been suggested as an explanation for sex differences in the rat (Kudo et al. 2002) and low elimination rates in humans (Andersen et al. 2006). The purpose of the present study was to estimate the serum elimination half-life of PFOS, PFHS, and PFOA in humans through the long-term follow-up of retired fluoro-chemical production workers. Although these retirees were no longer occupationally exposed, their serum concentrations were expected to be considerably higher than those of the general population.

Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the protein's ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced Salmonella invasion of colonic epithelial cells to levels associated with non-invasive deletion mutants. This study indicates that hmLF glycosylation is tightly regulated by gene expression and that glyco-variation is involved in modulating pathogen association.