The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
David C. Muddiman - One of the best experts on this subject based on the ideXlab platform.
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understanding the role of proteolytic digestion on discovery and targeted proteomic measurements using liquid chromatography tandem mass spectrometry and design of experiments
Journal of Proteome Research, 2013Co-Authors: Philip L Loziuk, Vincent L Chiang, Jack P. Wang, Ronald R. Sederoff, Quanzi Li, David C. MuddimanAbstract:Workflows in bottom-up proteomics have traditionally implemented the use of proteolysis during sample preparation; enzymatic digestion is most commonly performed using trypsin. This results in the hydrolysis of peptide bonds forming tryptic peptides, which can then be subjected to LC–MS/MS analysis. While the structure, specificity, and kinetics of trypsin are well characterized, a lack of consensus and understanding has remained regarding fundamental parameters critical to obtaining optimal data from a proteomics experiment. These include the type of trypsin used, pH during digestion, incubation temperature as well as enzyme-to-substrate ratio. Through the use of design of experiments (DOE), we optimized these parameters, resulting in deeper proteome coverage and a greater dynamic range of measurement. The knowledge gained from optimization of a discovery-based proteomics experiment was applied to targeted LC–MS/MS experiments using Protein Cleavage-isotope dilution mass spectrometry for absolute quantif...
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peptide production and decay rates affect the quantitative accuracy of Protein Cleavage isotope dilution mass spectrometry pc idms
Molecular & Cellular Proteomics, 2012Co-Authors: Christopher M. Shuford, Vincent L Chiang, Ronald R. Sederoff, David C. MuddimanAbstract:No consensus has been reached on the proper time to add stable-isotope labeled (SIL) peptides in Protein Cleavage isotope dilution mass spectrometry workflows. While quantifying 24 monolignol pathway enzymes in the xylem tissue of Populus trichocarpa, we compared the Protein concentrations obtained when adding the SIL standard peptides concurrently with the enzyme or after quenching of the digestion (i.e. postdigestion) and observed discrepancies for nearly all tryptic peptides investigated. In some cases, greater than 30-fold differences were observed. To explain these differences and potentially correct for them, we developed a mathematical model based on pseudo-first-order kinetics to account for the dynamic production and decay (e.g. degradation and precipitation) of the native peptide targets in conjunction with the decay of the SIL peptide standards. A time course study of the digests confirmed the results predicted by the proposed model and revealed that the discrepancy between concurrent and postdigestion introduction of the SIL standards was related to differential decay experienced by the SIL peptide and the native peptide in each method. Given these results, we propose concurrent introduction of the SIL peptide is most appropriate, though not free from bias. Mathematical modeling of this method reveals that overestimation of Protein quantities would still result when rapid peptide decay occurs and that this bias would be further exaggerated by slow proteolysis. We derive a simple equation to estimate the bias for each peptide based on the relative rates of production and decay. According to this equation, nearly half of the peptides evaluated here were estimated to have quantitative errors greater than 10% and in a few cases over 100%. We conclude that the instability of peptides can often significantly bias the Protein quantities measured in Protein Cleavage isotope dilution mass spectrometry-based assays and suggest peptide stability be made a priority when selecting peptides to use for quantification.
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Comprehensive quantification of monolignol-pathway enzymes in Populus trichocarpa by Protein Cleavage isotope dilution mass spectrometry.
Journal of Proteome Research, 2012Co-Authors: Christopher M. Shuford, Vincent L Chiang, Jack P. Wang, Ronald R. Sederoff, Ying-hsuan Sun, Hsi-chuan Chen, Rui Shi, David C. MuddimanAbstract:The economic value of wood/pulp from many tree species is largely dictated by the quantity and chemical properties of lignin, which is directly related to the composition and linkages of monolignols comprising the polymer. Although much is known regarding the monolignol biosynthetic pathway, our understanding is still deficient due to the lack of quantitative information at the proteomic level. We developed an assay based on Protein Cleavage isotope dilution mass spectrometry (PC-IDMS) for the determination of all potential, primary enzymes involved in the biosynthesis of monolignols and the peroxidases responsible for their polymerization to form lignin in the model tree species, Populus trichocarpa. Described is the identification of quantitative surrogate peptides through shotgun analysis of native and recombinant Proteins, optimization of trypsin proteolysis using fractional factorial design of experiments, and development of a liquid chromatography-selected reaction monitoring method for specific detection of all targeted peptides. Of the 25 targeted enzymes, three were undetected in the normal xylem tissues, and all but two of the detectable species showed good day-to-day precision (CV < 10%). This represents the most comprehensive assay for quantification of Proteins regulating monolignol biosynthesis and will lead to a better understanding of lignin formation at a systems level.
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absolute quantification of the model biomarker prostate specific antigen in serum by lc ms ms using Protein Cleavage and isotope dilution mass spectrometry
Journal of Proteome Research, 2004Co-Authors: David R Barnidge, Marcia K Goodmanson, George G Klee, David C. MuddimanAbstract:Protein Cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify Proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC−MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC−MS/MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of −0.003 (SE = 0.022), and R2 of 0.971. Reco...
Poole, Alastair W. - One of the best experts on this subject based on the ideXlab platform.
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Survival Protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and Protein Cleavage
'FASEB', 2016Co-Authors: Mattheij, Nadine J. A., Braun Attila, Castoldi Elisabetta, Pircher Joachim, Baaten, Constance C. F. M. J., Wülling Manuela, Kuijpers, Marijke J. E., Köhler Ralf, Van Kruchten Roger, Poole, Alastair W.Abstract:Scott syndrome is a rare bleeding disorder, characterized by altered Ca2+-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca2+-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P 0.05) with reduced PS exposure (−65 to 90%); 2) lowered Ca2+-dependent swelling (−80%) and membrane blebbing (−90%); 3) reduced calpain-dependent Protein Cleavage (−60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome.—Mattheij, N. J. A., Braun, A., van Kruchten, R., Castoldi, E., Pircher, J., Baaten, C. C. F. M. J., Wülling, M., Kuijpers, M. J. E., Köhler, R., Poole, A. W., Schreiber, R., Vortkamp, A., Collins, P. W., Nieswandt, B., Kunzelmann, K., Cosemans, J. M. E. M., Heemskerk, J. W. M. Survival Protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and Protein Cleavage
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Survival Protein anoctamin‐6 controls multiple platelet responses including phospholipid scrambling, swelling, and Protein Cleavage
'FASEB', 2016Co-Authors: Mattheij, Nadine J. A., Braun Attila, Kruchten Roger, Castoldi Elisabetta, Pircher Joachim, Baaten, Constance C. F. M. J., Wülling Manuela, Kuijpers, Marijke J. E., Köhler Ralf, Poole, Alastair W.Abstract:Scott syndrome is a rare bleeding disorder, characterized by altered Ca2+-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano) 6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca2+-dependent K(Ca)3.1Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6min (control 6.4min, P 0.05) with reduced PS exposure (265 to 90%); 2) lowered Ca2+-dependent swelling (280%) and membrane blebbing (-90%); 3) reduced calpain-dependent Protein Cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome
Siba K Samal - One of the best experts on this subject based on the ideXlab platform.
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mutations in the fusion Protein Cleavage site of avian paramyxovirus serotype 4 confer increased replication and syncytium formation in vitro but not increased replication and pathogenicity in chickens and ducks
PLOS ONE, 2013Co-Authors: Sa Xiao, Peter L Collins, Heather R Shive, Siba K SamalAbstract:To evaluate the role of the F Protein Cleavage site in the replication and pathogenicity of avian paramyxoviruses (APMVs), we constructed a reverse genetics system for recovery of infectious recombinant APMV-4 from cloned cDNA. The recovered recombinant APMV-4 resembled the biological virus in growth characteristics in vitro and in pathogenicity in vivo. The F Cleavage site sequence of APMV-4 (DIQPR↓F) contains a single basic amino acid, at the -1 position. Six mutant APMV-4 viruses were recovered in which the F Protein Cleavage site was mutated to contain increased numbers of basic amino acids or to mimic the naturally occurring Cleavage sites of several paramyxoviruses, including neurovirulent and avirulent strains of NDV. The presence of a glutamine residue at the -3 position was found to be important for mutant virus recovery. In addition, Cleavage sites containing the furin protease motif conferred increased replication and syncytium formation in vitro. However, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old and 2-week-old chickens, and 3-week-old ducks showed that none the F Protein Cleavage site mutations altered the replication, tropism, and pathogenicity of APMV-4, and no significant differences were observed among the parental and mutant APMV-4 viruses in vivo. Although parental and mutant viruses replicated somewhat better in ducks than in chickens, they all were highly restricted and avirulent in both species. These results suggested that the Cleavage site sequence of the F Protein is not a limiting determinant of APMV-4 pathogenicity in chickens and ducks.
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mutation of the f Protein Cleavage site of avian paramyxovirus type 7 results in furin Cleavage fusion promotion and increased replication in vitro but not increased replication tissue tropism or virulence in chickens
Journal of Virology, 2012Co-Authors: Sa Xiao, Sunil K Khattar, Madhuri Subbiah, Peter L Collins, Siba K SamalAbstract:ABSTRACT We constructed a reverse genetics system for avian paramyxovirus serotype 7 (APMV-7) to investigate the role of the fusion F glycoProtein in tissue tropism and virulence. The AMPV-7 F Protein has a single basic residue arginine (R) at position −1 in the F Cleavage site sequence and also is unusual in having alanine at position +2 (LPSSR↓FA) (underlining indicates the basic amino acids at the F Protein Cleavage site, and the arrow indicates the site of Cleavage.). APMV-7 does not form syncytia or plaques in cell culture, but its replication in vitro does not depend on, and is not increased by, added protease. Two mutants were successfully recovered in which the Cleavage site was modified to mimic sites that are found in virulent Newcastle disease virus isolates and to contain 4 or 5 basic residues as well as isoleucine in the +2 position: (RRQKR↓FI) or (RRKKR↓FI), named Fcs-4B or Fcs-5B, respectively. In cell culture, one of the mutants, Fcs-5B, formed protease-independent syncytia and grew to 10-fold-higher titers compared to the parent and Fcs-4B viruses. This indicated the importance of the single additional basic residue (K) at position −3. Syncytium formation and virus yield of the Fcs-5B virus was impaired by the furin inhibitor decanoyl-RVKR-CMK, whereas parental APMV-7 was not affected. APMV-7 is avirulent in chickens and is limited in tropism to the upper respiratory tract of 1-day-old and 2-week-old chickens, and these characteristics were unchanged for the two mutant viruses. Thus, the acquisition of furin cleavability by APMV-7 resulted in syncytium formation and increased virus yield in vitro but did not alter virus yield, tropism, or virulence in chickens.
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mutations in the fusion Protein Cleavage site of avian paramyxovirus serotype 2 increase cleavability and syncytium formation but do not increase viral virulence in chickens
Journal of Virology, 2011Co-Authors: Madhuri Subbiah, Sunil K Khattar, Peter L Collins, Siba K SamalAbstract:Avian paramyxovirus serotype 2 (APMV-2) is one of the nine serotypes of APMV, which infect a wide variety of avian species around the world. In this study, we constructed a reverse genetics system for recovery of infectious recombinant APMV-2 strain Yucaipa (APMV-2/Yuc) from cloned cDNA. The rescued recombinant virus (rAPMV-2) resembled the biological virus in growth properties in vitro and in pathogenicity in vivo. The reverse genetics system was used to analyze the role of the Cleavage site of the fusion (F) Protein in viral replication and pathogenesis. The Cleavage site of APMV-2/Yuc (KPASR↓F) contains only a single basic residue (position −1) that matches the preferred furin Cleavage site [RX(K/R)R↓]. (Underlining indicates the basic amino acids at the F Protein Cleavage site, and the arrow indicates the site of Cleavage.) Contrary to what would be expected for this Cleavage sequence, APMV-2 does not require, and is not augmented by, exogenous protease supplementation for growth in cell culture. However, it does not form syncytia, and the virus is avirulent in chickens. A total of 12 APMV-2 mutants with F Protein Cleavage site sequences derived from APMV serotypes 1 to 9 were generated. These sites contain from 1 to 5 basic residues. Whereas a number of these Cleavage sites are associated with protease dependence and lack of syncytium formation in their respective native viruses, when transferred into the APMV-2 backbone, all of them conferred protease independence, syncytium formation, and increased replication in cell culture. Examination of selected mutants during a pulse-chase experiment demonstrated an increase in F Protein Cleavage compared to that for wild-type APMV-2. Despite the gains in cleavability, replication, and syncytium formation, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old chicks, and 2-week-old chickens showed that the F Protein Cleavage site mutants did not exhibit increased pathogenicity and remained avirulent. These results imply that structural features in addition to the Cleavage site play a major role in the cleavability of the F Protein and the activity of the cleaved Protein. Furthermore, Cleavage of the F Protein is not a determinant of APMV-2 pathogenicity in chickens.
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complete genome sequences of avian paramyxovirus serotype 6 prototype strain hong kong and a recent novel strain from italy evidence for the existence of subgroups within the serotype
Virus Research, 2010Co-Authors: Sa Xiao, Sachin Kumar, Madhuri Subbiah, Peter L Collins, Roberta De Nardi, Calogero Terregino, Siba K SamalAbstract:Abstract Complete genome sequences were determined for two strains of avian paramyxovirus serotype 6 (APMV-6): the prototype Hong Kong (HK) strain and a more recent isolate from Italy (IT4524-2). The genome length of strain HK is 16236 nucleotide (nt), which is the same as for the other two APMV-6 strains (FE and TW) that have been reported to date, whereas that of strain IT4524-2 is 16230 nt. The length difference in strain IT4524-2 is due to a 6-nt deletion in the downstream untranslated region of the F gene. All of these viruses follow the “rule of six”. Each genome consists of seven genes in the order of 3′N-P-M-F-SH-HN-L5′, which differs from other APMV serotypes in containing an additional gene encoding the small hydrophobic (SH) Protein. Sequence comparisons revealed that strain IT4524-2 shares an unexpectedly low level of genome nt sequence identity (70%) and aggregate predicted amino acid (aa) sequence identity (79%) with other three strains, which in contrast are more closely related to each other with nt sequence 94–98% nt identity and 90–100% aggregate aa identity. Sequence analysis of the F-SH-HN genome region of two other recent Italian isolates showed that they fall in the HK/FE/TW group. The predicted signal peptide of IT4524-2 F Protein lacks the N-terminal first 10 aa that are present in the other five strains. Also, the F Protein Cleavage site of strain IT4524-2, R EP R ↓L, has two dibasic aa (arginine, R) compared to the monobasic F Protein Cleavage site of PEP R ↓L in the other strains. Reciprocal cross-hemagglutination inhibition (HI) assays using post-infection chicken sera indicated that strain IT4524-2 is antigenically related to the other APMV-6 strains, but with 4- to 8-fold lower HI tiers for the test sera between strain IT4524-2 and the other APMV-6 strains. Taken together, our results indicated that the APMV-6 strains represents a single serotype with two subgroups that differ substantially based on nt and aa sequences and can be distinguished by HI assay.
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role of fusion Protein Cleavage site in the virulence of newcastle disease virus
Microbial Pathogenesis, 2004Co-Authors: Aruna Panda, Zhuhui Huang, Subbiah Elankumaran, Daniel D Rockemann, Siba K SamalAbstract:Abstract Newcastle disease virus (NDV) causes a highly contagious and economically important disease in poultry. Viral determinants of NDV virulence are not completely understood. The amino acid sequence at the protease Cleavage site of the fusion (F) Protein has been postulated as a major determinant of NDV virulence. In this study, we have examined the role of F Protein Cleavage site sequence in NDV virulence using reverse genetics technology. The sequence G-R-Q-G-R present at the Cleavage site of the F Protein of avirulent strain LaSota was mutated to R-R-Q-K-R, which is present in the F Cleavage site of neurovirulent strain Beaudette C (BC). The resultant mutated LaSota V.F. virus did not require exogenous protease for infectivity in cell culture, indicating that the F Protein was cleaved by intracellular proteases. The virulence of the mutant and parental viruses was evaluated in vivo by intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index (IVPI) tests in chickens. Our results showed that the modification of the F Protein Cleavage site resulted in a dramatic increase in virulence from an ICPI value of 0.00 for LaSota to a value of 1.12 for LaSota V.F. However, the ICPI value of LaSota V.F. was lower than that of BC, which had a value of 1.58. Interestingly, the IVPI tests showed values of 0.00 for both LaSota and LaSota V.F. viruses, compared to the IVPI value of 1.45 of BC. In vitro characteristics of the viruses were also studied. Our results demonstrate that the efficiency of Cleavage of the F Protein plays an important role if the NDV is delivered directly into the brains of chicks, but there could be other viral factors that probably affect peripheral replication, viremia, or entry into the central nervous system.
Mattheij, Nadine J. A. - One of the best experts on this subject based on the ideXlab platform.
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Survival Protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and Protein Cleavage
'FASEB', 2016Co-Authors: Mattheij, Nadine J. A., Braun Attila, Castoldi Elisabetta, Pircher Joachim, Baaten, Constance C. F. M. J., Wülling Manuela, Kuijpers, Marijke J. E., Köhler Ralf, Van Kruchten Roger, Poole, Alastair W.Abstract:Scott syndrome is a rare bleeding disorder, characterized by altered Ca2+-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca2+-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P 0.05) with reduced PS exposure (−65 to 90%); 2) lowered Ca2+-dependent swelling (−80%) and membrane blebbing (−90%); 3) reduced calpain-dependent Protein Cleavage (−60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome.—Mattheij, N. J. A., Braun, A., van Kruchten, R., Castoldi, E., Pircher, J., Baaten, C. C. F. M. J., Wülling, M., Kuijpers, M. J. E., Köhler, R., Poole, A. W., Schreiber, R., Vortkamp, A., Collins, P. W., Nieswandt, B., Kunzelmann, K., Cosemans, J. M. E. M., Heemskerk, J. W. M. Survival Protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and Protein Cleavage
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Survival Protein anoctamin‐6 controls multiple platelet responses including phospholipid scrambling, swelling, and Protein Cleavage
'FASEB', 2016Co-Authors: Mattheij, Nadine J. A., Braun Attila, Kruchten Roger, Castoldi Elisabetta, Pircher Joachim, Baaten, Constance C. F. M. J., Wülling Manuela, Kuijpers, Marijke J. E., Köhler Ralf, Poole, Alastair W.Abstract:Scott syndrome is a rare bleeding disorder, characterized by altered Ca2+-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano) 6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca2+-dependent K(Ca)3.1Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6min (control 6.4min, P 0.05) with reduced PS exposure (265 to 90%); 2) lowered Ca2+-dependent swelling (280%) and membrane blebbing (-90%); 3) reduced calpain-dependent Protein Cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome
Vincent L Chiang - One of the best experts on this subject based on the ideXlab platform.
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understanding the role of proteolytic digestion on discovery and targeted proteomic measurements using liquid chromatography tandem mass spectrometry and design of experiments
Journal of Proteome Research, 2013Co-Authors: Philip L Loziuk, Vincent L Chiang, Jack P. Wang, Ronald R. Sederoff, Quanzi Li, David C. MuddimanAbstract:Workflows in bottom-up proteomics have traditionally implemented the use of proteolysis during sample preparation; enzymatic digestion is most commonly performed using trypsin. This results in the hydrolysis of peptide bonds forming tryptic peptides, which can then be subjected to LC–MS/MS analysis. While the structure, specificity, and kinetics of trypsin are well characterized, a lack of consensus and understanding has remained regarding fundamental parameters critical to obtaining optimal data from a proteomics experiment. These include the type of trypsin used, pH during digestion, incubation temperature as well as enzyme-to-substrate ratio. Through the use of design of experiments (DOE), we optimized these parameters, resulting in deeper proteome coverage and a greater dynamic range of measurement. The knowledge gained from optimization of a discovery-based proteomics experiment was applied to targeted LC–MS/MS experiments using Protein Cleavage-isotope dilution mass spectrometry for absolute quantif...
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peptide production and decay rates affect the quantitative accuracy of Protein Cleavage isotope dilution mass spectrometry pc idms
Molecular & Cellular Proteomics, 2012Co-Authors: Christopher M. Shuford, Vincent L Chiang, Ronald R. Sederoff, David C. MuddimanAbstract:No consensus has been reached on the proper time to add stable-isotope labeled (SIL) peptides in Protein Cleavage isotope dilution mass spectrometry workflows. While quantifying 24 monolignol pathway enzymes in the xylem tissue of Populus trichocarpa, we compared the Protein concentrations obtained when adding the SIL standard peptides concurrently with the enzyme or after quenching of the digestion (i.e. postdigestion) and observed discrepancies for nearly all tryptic peptides investigated. In some cases, greater than 30-fold differences were observed. To explain these differences and potentially correct for them, we developed a mathematical model based on pseudo-first-order kinetics to account for the dynamic production and decay (e.g. degradation and precipitation) of the native peptide targets in conjunction with the decay of the SIL peptide standards. A time course study of the digests confirmed the results predicted by the proposed model and revealed that the discrepancy between concurrent and postdigestion introduction of the SIL standards was related to differential decay experienced by the SIL peptide and the native peptide in each method. Given these results, we propose concurrent introduction of the SIL peptide is most appropriate, though not free from bias. Mathematical modeling of this method reveals that overestimation of Protein quantities would still result when rapid peptide decay occurs and that this bias would be further exaggerated by slow proteolysis. We derive a simple equation to estimate the bias for each peptide based on the relative rates of production and decay. According to this equation, nearly half of the peptides evaluated here were estimated to have quantitative errors greater than 10% and in a few cases over 100%. We conclude that the instability of peptides can often significantly bias the Protein quantities measured in Protein Cleavage isotope dilution mass spectrometry-based assays and suggest peptide stability be made a priority when selecting peptides to use for quantification.
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Comprehensive quantification of monolignol-pathway enzymes in Populus trichocarpa by Protein Cleavage isotope dilution mass spectrometry.
Journal of Proteome Research, 2012Co-Authors: Christopher M. Shuford, Vincent L Chiang, Jack P. Wang, Ronald R. Sederoff, Ying-hsuan Sun, Hsi-chuan Chen, Rui Shi, David C. MuddimanAbstract:The economic value of wood/pulp from many tree species is largely dictated by the quantity and chemical properties of lignin, which is directly related to the composition and linkages of monolignols comprising the polymer. Although much is known regarding the monolignol biosynthetic pathway, our understanding is still deficient due to the lack of quantitative information at the proteomic level. We developed an assay based on Protein Cleavage isotope dilution mass spectrometry (PC-IDMS) for the determination of all potential, primary enzymes involved in the biosynthesis of monolignols and the peroxidases responsible for their polymerization to form lignin in the model tree species, Populus trichocarpa. Described is the identification of quantitative surrogate peptides through shotgun analysis of native and recombinant Proteins, optimization of trypsin proteolysis using fractional factorial design of experiments, and development of a liquid chromatography-selected reaction monitoring method for specific detection of all targeted peptides. Of the 25 targeted enzymes, three were undetected in the normal xylem tissues, and all but two of the detectable species showed good day-to-day precision (CV < 10%). This represents the most comprehensive assay for quantification of Proteins regulating monolignol biosynthesis and will lead to a better understanding of lignin formation at a systems level.
