Further, the correlation between kallikrein expression and activity is complicated by the presence of at least one endogenous inhibitor (19, 21, 22). proteins. and purified as explained (11). Porcine trypsin and equine butyrylcholinesterase were purchased from Sigma. Urokinase plasminogen activator, tissue plasminogen activator, prostate-specific antigen, and bovine trypsin were purchased from Calbiochem. Preparation and Digestion of Proteomes. Generally, proteomes were labeled with ABPs at concentrations of 1C10 M for 5C60 min. After labeling, proteomes were denatured by adding 1 volume of 12 M urea (prepared at 50C to allow for total dissolution of the urea) and 1/50th volume Meisoindigo of DTT from a fresh 1 M stock. The combination was then heated to 65C for 15 min. Iodoacetamide was added to 40 mM from a fresh 1 M answer, and the solution was incubated at 37C for 30 min. The buffer was exchanged (10 mM ammonium bicarbonte/2 M urea) by gravity circulation gel filtration to remove extra reagents and unreacted probe, and reduce the urea concentration to a suitable concentration for trypsin activity. Gel filtration of small volume samples, for CE-only analysis, was performed by using Sephadex G-25 superfine resin (Amersham Pharmacia Biosciences) in custom 96-well filter plates from Innovative Microplate (Chicopee, MA). Larger volume samples to be analyzed by MS were gel-filtered using Econo-Pac 10-DG columns (Bio-Rad). After gel filtration, the samples were treated with 150 ng of trypsin (sequencing grade altered trypsin, Promega) per 10 l of sample for 1 h at 37C. Digests were stopped and prepared for CE by adding 200 mM citrate and 10% Triton X-100 at 1/20th of the sample volume. Preparation of Internal Standard Proteins. Bovine trypsin, porcine trypsin, and porcine elastase were diluted to 0.25 mg/ml in reaction buffer and labeled with 20 M fluorophosphonate polyethylene glycol (FP-PEG)-4,4-dif luoro-5,7-dimethyl-4-bora-3a,4a-diaza-for additional details). Identification of Probe-Labeled Peptides. LC-MS/MS data were searched against protein databases by using the sequest algorithm. Several modifications were launched to facilitate the interpretation of the sequest results (observe and Furniture 3 and 4, which are published as supporting information around the PNAS web site). Peak migration time CV’s ranged from 0.02% to 0.1%, whereas peak height and area reproducibility were between 15% and 30%. The extremely low variability in migration time and the high separation efficiencies are among the best reported for peptide analysis by CE (12), and are particularly noteworthy, considering the presence of problematic background proteomes such as undiluted plasma. To assess the sensitivity of this analysis method, known amounts of a purified serine hydrolase (butyrylcholinesterase) were added to mouse heart proteome before labeling with 10 M FP-PEG-TAMRA (Fig. 1Protein Reference Tissue distribution Molecular mass, kDa Candidate tumor suppressor OVCA2 “type”:”entrez-protein”,”attrs”:”text”:”Q9D7E3″,”term_id”:”81881544″,”term_text”:”Q9D7E3″Q9D7E3 ? 24.2 Lysophospholipase 2 “type”:”entrez-protein”,”attrs”:”text”:”Q9WTL7″,”term_id”:”41017420″,”term_text”:”Q9WTL7″Q9WTL7 ? 24.8 Monoglyceride lipase “type”:”entrez-protein”,”attrs”:”text”:”O35678″,”term_id”:”47117040″,”term_text”:”O35678″O35678 ? 33.4 Kynurenine formamidase “type”:”entrez-protein”,”attrs”:”text”:”Q8K4H1″,”term_id”:”81878680″,”term_text”:”Q8K4H1″Q8K4H1 ? 34.2 Carboxylesterase ML1 “type”:”entrez-protein”,”attrs”:”text”:”Q924V7″,”term_id”:”81902695″,”term_text”:”Q924V7″Q924V7 ? 41.5 Much like platelet activating factor acetylhydrolase “type”:”entrez-protein”,”attrs”:”text”:”Q8VDG7″,”term_id”:”341941227″,”term_text”:”Q8VDG7″Q8VDG7 ? 43.5 Arylacetamide deacetylase “type”:”entrez-protein”,”attrs”:”text”:”Q8VCF2″,”term_id”:”57012612″Q8VCF2 ? 45.3 Putative carboxylesterase “type”:”entrez-protein”,”attrs”:”text”:”Q91XD5″,”term_id”:”81902393″,”term_text”:”Q91XD5″Q91XD5 ? 58.2 Liver carboxylesterase ESTM_MOUSE ? 61.5 Much like carboxylesterase 2 “type”:”entrez-protein”,”attrs”:”text”:”Q8QZR3″,”term_id”:”81914815″,”term_text”:”Q8QZR3″Q8QZR3 ? 61.9 Putative carboxylesterase “type”:”entrez-protein”,”attrs”:”text”:”Q8BK48″,”term_id”:”81913291″,”term_text”:”Q8BK48″Q8BK48 ? 62.3 Carboxylesterase 1 “type”:”entrez-protein”,”attrs”:”text”:”Q8VCC2″,”term_id”:”51339201″,”term_text”:”Q8VCC2″Q8VCC2 ? 62.7 Cholinesterase CHLE_MOUSE ? 68.5 Prolyl-oligopeptidase (putative) “type”:”entrez-protein”,”attrs”:”text”:”Q8BKS6″,”term_id”:”81896523″,”term_text”:”Q8BKS6″Q8BKS6 ? 75.9 Prolyl endopeptidase PPCE_MOUSE ? 80.8 Dipeptidyl peptidase IV DPP4_MOUSE ? 87.4 Tripeptidyl-peptidase II TPP2_MOUSE ? 140.0 Lysophospholipase “type”:”entrez-protein”,”attrs”:”text”:”Q8BWM6″,”term_id”:”41017296″Q8BWM6 ?, ? 22.8 Putative lipase “type”:”entrez-protein”,”attrs”:”text”:”Q9DB29″,”term_id”:”81906062″,”term_text”:”Q9DB29″Q9DB29 ?, ? 28.0 Esterase 10 “type”:”entrez-protein”,”attrs”:”text”:”Q9R0P3″,”term_id”:”62900337″,”term_text”:”Q9R0P3″Q9R0P3 ?, ? 31.3 WilliamsBeuren syndrome critical region 21 “type”:”entrez-protein”,”attrs”:”text”:”Q8K4F5″,”term_id”:”81878678″,”term_text”:”Q8K4F5″Q8K4F5 ?, ? 33.6 Meisoindigo Platelet-activating factor acetylhydrolase PAFA_MOUSE ?, ? 49.4 Liver carboxylesterase 22 ES22_MOUSE ?, ? 61.6 Carboxylesterase 3 “type”:”entrez-protein”,”attrs”:”text”:”Q8VCT4″,”term_id”:”57013389″,”term_text”:”Q8VCT4″Q8VCT4 ?, ? 61.8 Putative carboxylesterase “type”:”entrez-protein”,”attrs”:”text”:”Q8R097″,”term_id”:”81914833″,”term_text”:”Q8R097″Q8R097 ?, ? 62.7 Bile-salt-activated lipase BAL_MOUSE ?, ? 65.8 Much like and 7, which is published as supporting information around the PNAS web site). This analysis confirmed Meisoindigo that this CE-LIF separation.These efforts have been complicated by the close sequence homology among users, overlapping tissue distributions, and overlapping substrate selectivities. were purchased from Sigma. Urokinase plasminogen activator, tissue plasminogen activator, prostate-specific antigen, and bovine trypsin were purchased from Calbiochem. Preparation and Digestion of Proteomes. Generally, proteomes were labeled with ABPs at concentrations of 1C10 M for 5C60 min. After labeling, proteomes were denatured by adding 1 volume of 12 M urea (prepared at 50C to allow for total dissolution of the urea) and 1/50th volume of DTT from a fresh 1 M stock. The combination was then heated to 65C for 15 min. Iodoacetamide was added to 40 mM from a fresh 1 M answer, and the solution was incubated at 37C for 30 min. The buffer was exchanged (10 mM ammonium bicarbonte/2 M urea) by gravity circulation gel filtration to remove extra reagents and unreacted probe, and reduce the urea concentration to a suitable concentration for trypsin activity. Gel filtration of small volume samples, for CE-only analysis, was performed by using Sephadex G-25 superfine resin (Amersham Pharmacia Biosciences) in custom 96-well filter plates from Innovative Microplate (Chicopee, MA). Larger volume samples to be analyzed by MS were gel-filtered using Econo-Pac 10-DG columns (Bio-Rad). After gel filtration, the samples were treated with 150 ng of trypsin (sequencing grade altered trypsin, Promega) per 10 l of sample for 1 h at 37C. Digests were Meisoindigo stopped and prepared for CE by adding 200 mM citrate and 10% Triton X-100 at 1/20th of the sample volume. Preparation of Internal Standard Proteins. Bovine trypsin, porcine trypsin, and porcine elastase were diluted to 0.25 mg/ml in reaction buffer and labeled with 20 M fluorophosphonate polyethylene glycol (FP-PEG)-4,4-dif luoro-5,7-dimethyl-4-bora-3a,4a-diaza-for additional details). Identification of Probe-Labeled Peptides. LC-MS/MS data were searched against protein databases by using the sequest algorithm. Several modifications were launched to facilitate the interpretation of the sequest results (observe and Furniture 3 and 4, which are published as supporting information around the PNAS web site). Peak migration time CV’s ranged from 0.02% to 0.1%, whereas peak height and area reproducibility were between 15% and 30%. The extremely low variability in migration time and the high separation efficiencies are among the best reported for peptide analysis by CE (12), and are particularly noteworthy, considering the presence of problematic background proteomes such as undiluted plasma. To assess the sensitivity of this analysis method, known amounts of a purified serine hydrolase (butyrylcholinesterase) were added to mouse heart proteome before labeling with 10 M FP-PEG-TAMRA (Fig. 1Protein Reference Tissue distribution Molecular mass, kDa Candidate tumor suppressor OVCA2 “type”:”entrez-protein”,”attrs”:”text”:”Q9D7E3″,”term_id”:”81881544″,”term_text”:”Q9D7E3″Q9D7E3 ? 24.2 Lysophospholipase 2 “type”:”entrez-protein”,”attrs”:”text”:”Q9WTL7″,”term_id”:”41017420″,”term_text”:”Q9WTL7″Q9WTL7 ? 24.8 Monoglyceride lipase “type”:”entrez-protein”,”attrs”:”text”:”O35678″,”term_id”:”47117040″,”term_text”:”O35678″O35678 ? 33.4 Kynurenine formamidase “type”:”entrez-protein”,”attrs”:”text”:”Q8K4H1″,”term_id”:”81878680″,”term_text”:”Q8K4H1″Q8K4H1 ? 34.2 Carboxylesterase ML1 “type”:”entrez-protein”,”attrs”:”text”:”Q924V7″,”term_id”:”81902695″,”term_text”:”Q924V7″Q924V7 ? 41.5 Much like platelet activating factor acetylhydrolase “type”:”entrez-protein”,”attrs”:”text”:”Q8VDG7″,”term_id”:”341941227″,”term_text”:”Q8VDG7″Q8VDG7 ? 43.5 Arylacetamide Rabbit Polyclonal to RRAGB deacetylase “type”:”entrez-protein”,”attrs”:”text”:”Q8VCF2″,”term_id”:”57012612″Q8VCF2 ? 45.3 Putative carboxylesterase “type”:”entrez-protein”,”attrs”:”text”:”Q91XD5″,”term_id”:”81902393″,”term_text”:”Q91XD5″Q91XD5 ? 58.2 Liver carboxylesterase ESTM_MOUSE ? 61.5 Much like carboxylesterase 2 “type”:”entrez-protein”,”attrs”:”text”:”Q8QZR3″,”term_id”:”81914815″,”term_text”:”Q8QZR3″Q8QZR3 ? 61.9 Putative carboxylesterase “type”:”entrez-protein”,”attrs”:”text”:”Q8BK48″,”term_id”:”81913291″,”term_text”:”Q8BK48″Q8BK48 ? 62.3 Carboxylesterase 1 “type”:”entrez-protein”,”attrs”:”text”:”Q8VCC2″,”term_id”:”51339201″,”term_text”:”Q8VCC2″Q8VCC2 ? 62.7 Cholinesterase CHLE_MOUSE ? 68.5 Prolyl-oligopeptidase (putative) “type”:”entrez-protein”,”attrs”:”text”:”Q8BKS6″,”term_id”:”81896523″,”term_text”:”Q8BKS6″Q8BKS6 ? 75.9 Prolyl endopeptidase PPCE_MOUSE ? 80.8 Dipeptidyl peptidase IV DPP4_MOUSE ? 87.4 Tripeptidyl-peptidase II TPP2_MOUSE ? 140.0 Lysophospholipase “type”:”entrez-protein”,”attrs”:”text”:”Q8BWM6″,”term_id”:”41017296″Q8BWM6 ?, ? 22.8 Putative lipase “type”:”entrez-protein”,”attrs”:”text”:”Q9DB29″,”term_id”:”81906062″,”term_text”:”Q9DB29″Q9DB29 ?, ? 28.0 Esterase 10 “type”:”entrez-protein”,”attrs”:”text”:”Q9R0P3″,”term_id”:”62900337″,”term_text”:”Q9R0P3″Q9R0P3 ?, ? 31.3 WilliamsBeuren syndrome critical region 21 “type”:”entrez-protein”,”attrs”:”text”:”Q8K4F5″,”term_id”:”81878678″,”term_text”:”Q8K4F5″Q8K4F5 ?, ? 33.6 Platelet-activating factor acetylhydrolase PAFA_MOUSE ?, ? 49.4 Liver carboxylesterase 22 ES22_MOUSE ?, ? 61.6 Carboxylesterase 3 “type”:”entrez-protein”,”attrs”:”text”:”Q8VCT4″,”term_id”:”57013389″,”term_text”:”Q8VCT4″Q8VCT4 ?, ? 61.8 Putative carboxylesterase “type”:”entrez-protein”,”attrs”:”text”:”Q8R097″,”term_id”:”81914833″,”term_text”:”Q8R097″Q8R097 ?, ? 62.7 Bile-salt-activated lipase BAL_MOUSE ?, ? 65.8 Much like and 7, which is published as supporting information around the PNAS web site). This analysis confirmed that this CE-LIF separation had successfully separated the majority of the kallikrein enzymes present in this sample, despite primary sequence identity between these enzymes of up to 96%. To demonstrate the power of the Xsite platform for quick and.
