Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Richard K. Niles is active.

Publication


Featured researches published by Richard K. Niles.


Nature Biotechnology | 2009

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.

Terri Addona; Susan E. Abbatiello; Birgit Schilling; Steven J. Skates; D. R. Mani; David M. Bunk; Clifford H. Spiegelman; Lisa J. Zimmerman; Amy-Joan L. Ham; Hasmik Keshishian; Steven C. Hall; Simon Allen; Ronald K. Blackman; Christoph H. Borchers; Charles Buck; Michael P. Cusack; Nathan G. Dodder; Bradford W. Gibson; Jason M. Held; Tara Hiltke; Angela M. Jackson; Eric B. Johansen; Christopher R. Kinsinger; Jing Li; Mehdi Mesri; Thomas A. Neubert; Richard K. Niles; Trenton Pulsipher; David F. Ransohoff; Henry Rodriguez

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low μg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.


Journal of Proteome Research | 2008

The Proteomes of Human Parotid and Submandibular/Sublingual Gland Salivas Collected as the Ductal Secretions

Paul C. Denny; Fred K. Hagen; Markus Hardt; Lujian Liao; Weihong Yan; Martha Arellanno; Sara Bassilian; Gurrinder S. Bedi; Pinmannee Boontheung; Daniel Cociorva; Claire Delahunty; Trish Denny; Jason Dunsmore; Kym F. Faull; Joyce Gilligan; Mireya Gonzalez-Begne; Frédéric Halgand; Steven C. Hall; Xuemei Han; Bradley S. Henson; Johannes A. Hewel; Shen Hu; Sherry Jeffrey; Jiang Jiang; Joseph A. Loo; Rachel R. Ogorzalek Loo; Daniel Malamud; James E. Melvin; Olga Miroshnychenko; Mahvash Navazesh

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.


Analytical Biochemistry | 2011

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma

Penelope M. Drake; Birgit Schilling; Richard K. Niles; Miles Braten; Eric B. Johansen; Haichuan Liu; Michael T. Lerch; Dylan J. Sorensen; Bensheng Li; Simon Allen; Steven C. Hall; H. Ewa Witkowska; Fred E. Regnier; Bradford W. Gibson; Susan J. Fisher

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.


PLOS ONE | 2009

Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

Shoba Subramanian; Markus Hardt; Youngchool Choe; Richard K. Niles; Eric B. Johansen; Jennifer Legac; Jiri Gut; Iain D. Kerr; Charles S. Craik; Philip J. Rosenthal

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P1 – P4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P2 position. Second, with overlapping peptides spanning α and β globin and proteolysis-dependent 18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.


Analytical Chemistry | 2009

Acid-Catalyzed Oxygen-18 Labeling of Peptides

Richard K. Niles; H. Ewa Witkowska; Simon Allen; Steven C. Hall; Susan J. Fisher; Markus Hardt

In enzymatic (18)O-labeling strategies for quantitative proteomics, the exchange of carboxyl oxygens at low pH is a common, undesired side reaction. We asked if acid-catalyzed back exchange could interfere with quantitation and whether the reaction itself could be used as method for introducing (18)O label into peptides. Several synthetic peptides were dissolved in dilute acid containing 50% (v/v) H(2)(18)O and incubated at room temperature. Aliquots were removed over a period of 3 weeks and analyzed by tandem mass spectrometry (MS/MS). (18)O-incorporation ratios were determined by linear regression analysis that allowed for multiple stable-isotope incorporations. At low pH, peptides exchanged their carboxyl oxygen atoms with the aqueous solvent. The isotope patterns gradually shifted to higher masses until they reached the expected binomial distribution at equilibrium after approximately 11 days. Reaction rates were residue- and sequence-specific. Due to its slow nature, the acid-catalyzed back exchange is expected to minimally interfere with enzymatic (18)O-labeling studies provided that storage and analysis conditions minimize low-pH exposure times. On its own, acid-catalyzed (18)O labeling is a general tagging strategy that is an alternative to the chemical, metabolic, and enzymatic isotope-labeling schemes currently used in quantitative proteomics.


PLOS ONE | 2012

Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins.

Nancy J. Phillips; Christopher T. Steichen; Birgit Schilling; Deborah M. B. Post; Richard K. Niles; Thomas B. Bair; Megan L. Falsetta; Michael A. Apicella; Bradford W. Gibson

Neisseria gonorrhoeae, the causative agent of gonorrhea, can form biofilms in vitro and in vivo. In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with 13C6-arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values <0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related to this shift may have other functions.


Journal of Proteome Research | 2012

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers.

Penelope M. Drake; Birgit Schilling; Richard K. Niles; Akraporn Prakobphol; Bensheng Li; Kwanyoung Jung; Wonryeon Cho; Miles Braten; Halina D. Inerowicz; Katherine E. Williams; Matthew E. Albertolle; Jason M. Held; Demetris C. Iacovides; Dylan J. Sorensen; Obi L. Griffith; Eric B. Johansen; Anna M. Zawadzka; Michael P. Cusack; Simon Allen; Matthew Gormley; Steven C. Hall; H. Ewa Witkowska; Joe W. Gray; Fred E. Regnier; Bradford W. Gibson; Susan J. Fisher

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ∼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.


Proteomics | 2008

A mass spectrometry-based strategy for detecting and characterizing endogenous proteinase activities in complex biological samples.

Sarah Robinson; Richard K. Niles; H. Ewa Witkowska; Kirsten J. Rittenbach; Robert J. Nichols; Jonathan A. Sargent; Scott E. Dixon; Akraporn Prakobphol; Steven C. Hall; Susan J. Fisher; Markus Hardt

Endogenous proteinases in biological fluids such as human saliva produce a rich peptide repertoire that reflects a unique combination of enzymes, substrates, and inhibitors/activators. Accordingly, this subproteome is an interesting source of biomarkers for disease processes that either directly or indirectly involve proteolysis. However, the relevant proteinases, typically very low abundance molecules, are difficult to classify and identify. We hypothesized that a sensitive technique for monitoring accumulated peptide products in an unbiased, global manner would be very useful for detecting and profiling proteolytic activities in complex biological samples. Building on the longstanding use of 18O isotope‐based approaches for the classification of proteolytic and other enzymatic processes we devised a new method for evaluating endogenous proteinases. Specifically, we showed that upon ex vivo incubation endogenous proteinases in human parotid saliva introduced 18O from isotopically enriched water into the C‐terminal carboxylic groups of their peptide products. Subsequent peptide sequence determination and inhibitor profiling enabled the detection of discrete subsets of proteolytic products that were generated by different enzymes. As a proof‐of‐principle we used one of these fingerprints to identify the relevant activity as tissue kallikrein. We termed this technique PALeO. Our results suggest that PALeO is a rapid and highly sensitive method for globally assessing proteinase activities in complex biological samples.


American Journal of Physiology-lung Cellular and Molecular Physiology | 2013

Protein composition of bronchoalveolar lavage fluid and airway surface liquid from newborn pigs

Jennifer A. Bartlett; Matthew E. Albertolle; Christine L. Wohlford-Lenane; Alejandro A. Pezzulo; Joseph Zabner; Richard K. Niles; Susan J. Fisher; Paul B. McCray; Katherine E. Williams

The airway mucosa and the alveolar surface form dynamic interfaces between the lung and the external environment. The epithelial cells lining these barriers elaborate a thin liquid layer containing secreted peptides and proteins that contribute to host defense and other functions. The goal of this study was to develop and apply methods to define the proteome of porcine lung lining liquid, in part, by leveraging the wealth of information in the Sus scrofa database of Ensembl gene, transcript, and protein model predictions. We developed an optimized workflow for detection of secreted proteins in porcine bronchoalveolar lavage (BAL) fluid and in methacholine-induced tracheal secretions [airway surface liquid (ASL)]. We detected 674 and 3,858 unique porcine-specific proteins in BAL and ASL, respectively. This proteome was composed of proteins representing a diverse range of molecular classes and biological processes, including host defense, molecular transport, cell communication, cytoskeletal, and metabolic functions. Specifically, we detected a significant number of secreted proteins with known or predicted roles in innate and adaptive immunity, microbial killing, or other aspects of host defense. In greatly expanding the known proteome of the lung lining fluid in the pig, this study provides a valuable resource for future studies using this important animal model of pulmonary physiology and disease.


Journal of Proteomics | 2015

Urine, peritoneal fluid and omental fat proteomes of reproductive age women: Endometriosis-related changes and associations with endocrine disrupting chemicals

Katherine E. Williams; Olga Miroshnychenko; Eric B. Johansen; Richard K. Niles; Rajeshwari Sundaram; Kurunthachalam Kannan; Matthew E. Albertolle; Yan Zhou; Namrata Prasad; Penelope M. Drake; Linda C. Giudice; Steven C. Hall; H. Ewa Witkowska; Germaine M. Buck Louis; Susan J. Fisher

UNLABELLED Endometriosis, ectopic growth of the uterine lining (endometrium), which affects 6-11% of reproductive age women, is associated with pelvic pain and infertility. We investigated the peritoneal fluid (PF), urine and omental fat (OF) proteomes of women with endometriosis vs. individuals with no surgically visualized endometriosis. All participants were enrolled in the NICHD-funded ENDO Study. A two-step proteomic study was performed. The first, a broad survey, employed a semi-quantitative gel LC-mass spectrometry (MS) workflow: SDS PAGE fractionation, trypsin digestion and LC-MS/MS. The results showed sample integrity but failed to detect any differences between women with and without endometriosis. The second step was a quantitative analysis of OF samples. We employed another sample set (n=30) from women ± disease and isobaric mass-tag (iTRAQ) chemistry to label peptides and 2D LC-MS/MS for protein identification and quantification. Three proteins-matrix metalloproteinase-9, neutrophil elastase, and FAM49B-were significantly lower in abundance in samples from women with endometriosis. Interestingly, neutrophil elastase and FAM49B levels were associated with higher levels of a subset of endocrine disrupting chemicals (EDCs) that were previously measured in the same samples. The results of these experiments showed the feasibility of associating endometriosis with changes in the OF protein repertoire and EDC levels. BIOLOGICAL SIGNIFICANCE Endometriosis, pathological growth of the uterine lining, is associated with significant morbidities, including pain and infertility. However, the causes of this common condition are poorly understood. This study determined whether endometriosis was associated with changes in the protein composition of peritoneal fluid, urine and/or omental fat. A protein of unknown function (FAM49B) and two proteinases (metalloproteinase-9, neutrophil elastase) were down regulated in OF samples from women with versus without endometriosis. These findings suggested proteinase imbalances at sites that were distant from the endometriotic lesions. Additionally, FAM49B and neutrophil elastase levels were associated with higher levels of a subset of environmental chemicals that were quantified in the same samples, suggesting other possible associations. Thus, this work generated hypotheses that will be tested in further studies.

Collaboration


Dive into the Richard K. Niles's collaboration.

Top Co-Authors

Avatar

Steven C. Hall

University of California

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Birgit Schilling

Buck Institute for Research on Aging

View shared research outputs
Top Co-Authors

Avatar

Bradford W. Gibson

Buck Institute for Research on Aging

View shared research outputs
Top Co-Authors

Avatar

Simon Allen

University of California

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Researchain Logo
Decentralizing Knowledge