www.clinical.proteomics-journal.com
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Proteomics - Clinical Applications
Technical Brief Intraluminal Proteome and Peptidome of Human Urinary Extracellular Vesicles *Xinyu Liu1,2, *Clizia Chinello3, Luca Musante1, Marta Cazzaniga3, Dorota Tataruch1, Giulio Calzaferri1, Andrew James Smith3, Gabriele De Sio3, Fulvio Magni3, Hequn Zou2, Harry Holthofer1
1 2
Centre for BioAnalytical Sciences, Dublin City University, Dublin 9, Ireland Institute of Nephrology and Urology, The Third Affiliated Hospital of Southern Medical
University, Guangzhou, China 3
Department of Health Science, University of Milano-Bicocca Monza, Italy
*equally contributing authors Correspondence: Prof. Fulvio Magni, PhD, Department of Health Science, University of Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy Email: [email protected] Fax: +39 0264488252
Abbreviations: UEV, urinary extracellular vesicles; MS, mass spectrometry; THP, Tamm-Horsfall glycoprotein, uromodulin; RT, room temperature; MVB, multivesicular body; SN, supernatant; P,precipitate; MWCO, molecular weight cut-off; HD, hydrostatic dialysis; HDa, hydrostatic dialysis above 1000kDa; CaCl2, calcium chloride; TCEP, TRIS (carboxyethyl) phosphine; IAA, iodoacetamide; DOC, sodium deoxycholate; NAC, N-acetylcysteine; FA, formic acid; TEM, transmission electron microscope; TSG101, tumor susceptibility gene 101; ESCRT, endosomal sorting complex required for transport Keywords: Urinary extracellular vesicles/ Tamm-Horsfall glycoprotein/ Proteome/ Peptidome/ Exosome Total number of words: 2604 from abstract to legend of Table 1
Received: 19-Aug-2014; Revised: 28-Oct-2014; Accepted: 26-Nov-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/prca.201400085. This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 2
Proteomics - Clinical Applications
Statement of clinical relevance Urinary Extracellular Vesicles (UEV) emerge as a unique source for clinically relevant biomarkers of disease activity (both systemic and kidney diseases) while a systematic approach to define an optimized protocol for UEV isolation has been lacking. The simplified protocol for isolation as described here meets the needs of a clinical laboratory for a simple and fast procedure of sample preparation before detailed analysis by Mass-Spectrometry (MS). Of particular importance is the possibility to study not only the endoproteome but also endopeptidome of UEV thus greatly facilitating biomarker discovery. Abstract Purpose: Urinary extracellular vesicles (UEV) are a novel source for disease biomarker discovery. However, Tamm-Horsfall glycoprotein (THP) is still a challenge for proteomic analysis since it can inhibit detection of low abundance proteins. Here we introduce a new approach which doesn’t involve an ultracentrifugation step to enrich vesicles and
which
reduces the amount of THP to manageable levels. Experimental design: UEV were dialyzed and ultrafiltered after reduction and alkylation. The retained fraction was digested with trypsin to reduce the remaining THP and incubated with sodium deoxycholate. The internal peptidome and the internal proteome were analyzed by LC-ESI-MS. Results: 942 different proteins and 3115 unique endogenous peptide fragments deriving from 973 different protein isoforms were identified. Around 82% of the key Endosomal Sorting Complex Required for Transport (ESCRT) components of UEV generation could be detected from the intraluminal content. This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 3
Proteomics - Clinical Applications
Conclusions and clinical relevance: Our UEV preparation protocol provides a simplified way to investigate the intraluminal proteome and peptidome, in particular the subpopulation of UEV of the trypsin resistant class of exosomes (positive for Tumor susceptibility gene101) and eliminates the majority of interfering proteins like THP. This method allows the possibility to study endoproteome and endopeptidome of UEV thus greatly facilitating biomarker discovery.
Main text Urinary extracellular vesicles (UEV), include a heterogeneous population of vesicles such as exosomes, exosome-like vesicles and microvesicles [1]. UEV of different classes have different biogenesis, morphological features, and functions. Urinary exosomes mostly derive from the endosomal pathway and are released into the urine when the intracellular multivesicular body (MVB) fuses with the cellular plasma membrane [2]. Due to their biogenesis, they appear to mirror the internal events of the respective epithelial cells and are suitable as potential disease biomarkers [2-5]. However, Tamm-Horsfall protein (THP), an abundant urinary protein is a challenge for precise and deep proteomic analysis of the urinary vesicle contents since it can interfere with the detection of low abundance proteins [3]. Currently, the widely used method to isolate vesicles
relies on differential centrifugations, which enrich vesicles while resulting in high
amount of THP, even in presence of reducing agents [6]. Alternative methods have been proposed to enrich UEV without massive THP interference using e.g. sucrose cushion and/or gradient isolation procedures. All of them are time consuming and labor intensive with This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 4
Proteomics - Clinical Applications
multiple centrifugation steps, which are not suitable for a practical clinical analysis. The other UEV isolation approaches reported before, including filtration, immunoaffinity, or using vesicle precipitation reagents (ExoQuick-TC) also have their distinct limitations and not widely applied [4]. Here we introduce a new approach, named hydrostatic dialysis (HD), to enrich vesicles which can easily process large amounts of urine without involving an ultracentrifugation step, thus making it more convenient and applicable for clinical use. Like traditional methods, our approach is similarly limited by remaining abundance of THP unless further processed. Thus, the main objective of our study was to decrease the THP interference by introducing trypsin digestion, while preserving the intraluminal proteome and peptidome. In our method the first morning void urine samples were collected from healthy volunteers (3 males and 2 females, age 29-43 years). No history of kidney or other general disease of any of the individuals, and no medications or alcohol was taken 12 hours prior to urine collection. No protease inhibitors or other urinary preservatives were added to the urine samples. Informed consent was obtained from all the volunteers before sample donation. The protocol was approved by the ethical committee of Dublin City University. Figure 1 shows the workflow for UEV enrichment and characterization. Briefly, 4 liters of crude urine was centrifuged at 2000g for 30 min at room temperature (RT) [7]. Supernatant (SN) was poured into a funnel connected to the 1,000kDa molecular weight cut-off (MWCO) membrane. Soluble proteins below the MWCO, yellow pigments and salts could be effectively removed from urine within around 6 hours. After washing with 200ml milliQ water, the remaining 6-8ml solution retained inside the dialysis tube (hydrostatic This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 5
Proteomics - Clinical Applications
dialysis above 1000kDa, HDa) was collected. The procedure for processing 0.5 l of urine with one HD system takes about 8 hours. Negative transmission electron microscopy (TEM) analysis was then performed to verify the presence of UEV. To eliminate the contamination of THP, HDa solution with 1mg protein (as determined by Bradford protein assay) was reduced by 10mM TRIS (carboxyethyl) phosphine (TCEP), 6M Urea, 0.1mM Ethyldiamminotetracetic acid (EDTA) and 0.2M Tris buffer pH8.8 in dark for 2 hours at RT, followed by alkylation with 20mM iodoacetamide (IAA) in dark for 2 hours. After that, 40mM N-acetyl cysteine (NAC) was added to the samples to quench the excess of IAA. Then the sample was ultrafiltrated with Vivaspin 15R ultrafiltration device having 10kDa MWCO membranes (Sartorius Stedim Biotech GmbH, Goettingen, Germany) to remove salts (10kDa), present in the HDa fraction and not belonging to UEV, and to reduce the remaining THP as much as possible. Then the sample was ultrafiltered by 30kDa MWCO Vivaspin 15R device to remove peptides. This step was repeated 3 times. The last retained fraction above 30kDa was incubated with 1% (w/v) sodium deoxycholate (DOC) to solubilize the membrane of UEV and release the intraluminal proteins which were reduced and alkylated as described above. Internal peptides were first isolated by ultrafiltration (10kDa) and then the retained solution (>10kDa) underwent three cycles of trypsin digestion with 1% DOC as previously described. The internal peptidome This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 6
Proteomics - Clinical Applications
(below 10 kDa) and internal proteome (below 30 kDa) were then acidified with 1% (v/v) formic acid (FA) to precipitate DOC and then the supernatant cleaned up by Sep-Pak C18 cartridge (Waters Associates, Milford, MA) as well [8].TEM analysis showed a heterogeneous population of nanoparticles in HDa fraction (Figure 1B). The size range (between 30 and 300 nm diameter) is consistent with that of exosomes, exosome-like and microvesicles. Each fraction during the UEV processing step of the workflow
was analyzed
by SDS-PAGE and Western blotting (Figure 1 C, D, E). After reduction, alkylation (lane 2) and digestion (lanes 3-5), the amount of intact THP (Figure 1C and E) in HDa fraction was strongly reduced. The intensive signal of Tumor susceptibility gene 101 (TSG101)(~ 46 kDa) (Figure 1 D) used as a exosome marker, was visible at the initial vesicles fractions (HDa, lane 1), suggesting the effective enrichment of UEV with our isolation method and after the trypsin digestion which did not affect the integrity of TSG101 (lane 2-5). Conversely, it disappeared after DOC treatment and trypsin digestion (lane 6). This indicates that, only after solubilising the phospholipidic bilayer by DOC, the trypsin can digest TSG101. The above results demonstrate the feasibility and efficiency of our approach, which uses trypsin digestion and ultrafiltration, to strongly reduce the interference of THP while fully preserving the integrity of (TSG101 positive) exosomes. As expected, the contaminant proteins outside UEV, particularly the THP, were cleaved into small peptides and removed by consequent 30kDa MWCO ultrafiltration. Previous studies demonstrated formidable resistance of UEV to various treatments [7], but no previous study has systematically considered the structural stability after reduction, alkylation and trypsin digestion. The Western blot results clearly indicate the structural This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 7
Proteomics - Clinical Applications
integrity of such vesicles as even 3 consecutive trypsinizations did not affect TSG101 signal. Thus, this feature also give us the opportunity to separate and purify trypsin resistant UEV, mostly exosomes, by means of simple ultrafiltration. Next, the following DOC treatment, reduction, alkylation, trypsinization, and series of ultrafiltrations could further separate proteins and peptides inside the vesicles, which gives us a reliable endoproteome and endopeptidome mapping of the UEV involved. While the urinary proteome has been widely investigated earlier in several studies, the peptidome is less well known, and it represents an important source of potential biomarkers [9]. Taking advantage of the ultrafiltration step with different MWCO filters, we isolated and characterized, for the first time, the endopeptidome of the UEVs. Internal vesicular proteome and peptidome was identified by nLC-ESI-MS/MS [10]. Lyophilized, desalted fractions were re-suspended in 50µl of 0.1% TFA and the protein concentration was estimated using the NanoDrop spectrophotometer (Thermo Scientific, Bonn, Germany). For this, 5µg of protein sample was injected into Dionex UltiMate 3000 Rapid Separation LC nano system (Thermo Scientific) coupled with an Impact HDTM mass spectrometer (Bruker Daltonics, Bremen, Germany). Peptide samples were loaded onto a Pepmap pre-column (2cm x 100µm, 5µm, Thermo Scientific), followed by separation on 50cm Nano column (0.075mm, 2µm, Acclaim PepMap100, C18, Thermo Scientific), at a flow rate of 300nl/min. Multistep 360min gradients of acetonitrile were used. The column was connected to a nanoBoosterCaptiveSprayTM (Bruker Daltonics). Mass spectrometer was operated in data-dependent-acquisition mode, using the IDAS and RT2 functionalities. Peaklists were processed using an in-house Mascot search engine (v2.4.1). Database This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 8
Proteomics - Clinical Applications
searching was restricted to human reviewed database Swiss-Prot (accessed Apr 2014, 544,996 sequences; 193,815,432 residues). Mass tolerances were set at 20ppm for MS and 0.05Da for MS/MS. For digested samples (proteome), trypsin and Carbamidomethyl(C) were set as enzyme and as fixed modification, respectively. Automatic decoy database search and built-in Percolator algorithm were used to rescore search results and calculate posterior error probabilities for each peptide-spectrum match. All results were filtered to achieve global FDR
Page 1
Proteomics - Clinical Applications
Technical Brief Intraluminal Proteome and Peptidome of Human Urinary Extracellular Vesicles *Xinyu Liu1,2, *Clizia Chinello3, Luca Musante1, Marta Cazzaniga3, Dorota Tataruch1, Giulio Calzaferri1, Andrew James Smith3, Gabriele De Sio3, Fulvio Magni3, Hequn Zou2, Harry Holthofer1
1 2
Centre for BioAnalytical Sciences, Dublin City University, Dublin 9, Ireland Institute of Nephrology and Urology, The Third Affiliated Hospital of Southern Medical
University, Guangzhou, China 3
Department of Health Science, University of Milano-Bicocca Monza, Italy
*equally contributing authors Correspondence: Prof. Fulvio Magni, PhD, Department of Health Science, University of Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy Email: [email protected] Fax: +39 0264488252
Abbreviations: UEV, urinary extracellular vesicles; MS, mass spectrometry; THP, Tamm-Horsfall glycoprotein, uromodulin; RT, room temperature; MVB, multivesicular body; SN, supernatant; P,precipitate; MWCO, molecular weight cut-off; HD, hydrostatic dialysis; HDa, hydrostatic dialysis above 1000kDa; CaCl2, calcium chloride; TCEP, TRIS (carboxyethyl) phosphine; IAA, iodoacetamide; DOC, sodium deoxycholate; NAC, N-acetylcysteine; FA, formic acid; TEM, transmission electron microscope; TSG101, tumor susceptibility gene 101; ESCRT, endosomal sorting complex required for transport Keywords: Urinary extracellular vesicles/ Tamm-Horsfall glycoprotein/ Proteome/ Peptidome/ Exosome Total number of words: 2604 from abstract to legend of Table 1
Received: 19-Aug-2014; Revised: 28-Oct-2014; Accepted: 26-Nov-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/prca.201400085. This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 2
Proteomics - Clinical Applications
Statement of clinical relevance Urinary Extracellular Vesicles (UEV) emerge as a unique source for clinically relevant biomarkers of disease activity (both systemic and kidney diseases) while a systematic approach to define an optimized protocol for UEV isolation has been lacking. The simplified protocol for isolation as described here meets the needs of a clinical laboratory for a simple and fast procedure of sample preparation before detailed analysis by Mass-Spectrometry (MS). Of particular importance is the possibility to study not only the endoproteome but also endopeptidome of UEV thus greatly facilitating biomarker discovery. Abstract Purpose: Urinary extracellular vesicles (UEV) are a novel source for disease biomarker discovery. However, Tamm-Horsfall glycoprotein (THP) is still a challenge for proteomic analysis since it can inhibit detection of low abundance proteins. Here we introduce a new approach which doesn’t involve an ultracentrifugation step to enrich vesicles and
which
reduces the amount of THP to manageable levels. Experimental design: UEV were dialyzed and ultrafiltered after reduction and alkylation. The retained fraction was digested with trypsin to reduce the remaining THP and incubated with sodium deoxycholate. The internal peptidome and the internal proteome were analyzed by LC-ESI-MS. Results: 942 different proteins and 3115 unique endogenous peptide fragments deriving from 973 different protein isoforms were identified. Around 82% of the key Endosomal Sorting Complex Required for Transport (ESCRT) components of UEV generation could be detected from the intraluminal content. This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 3
Proteomics - Clinical Applications
Conclusions and clinical relevance: Our UEV preparation protocol provides a simplified way to investigate the intraluminal proteome and peptidome, in particular the subpopulation of UEV of the trypsin resistant class of exosomes (positive for Tumor susceptibility gene101) and eliminates the majority of interfering proteins like THP. This method allows the possibility to study endoproteome and endopeptidome of UEV thus greatly facilitating biomarker discovery.
Main text Urinary extracellular vesicles (UEV), include a heterogeneous population of vesicles such as exosomes, exosome-like vesicles and microvesicles [1]. UEV of different classes have different biogenesis, morphological features, and functions. Urinary exosomes mostly derive from the endosomal pathway and are released into the urine when the intracellular multivesicular body (MVB) fuses with the cellular plasma membrane [2]. Due to their biogenesis, they appear to mirror the internal events of the respective epithelial cells and are suitable as potential disease biomarkers [2-5]. However, Tamm-Horsfall protein (THP), an abundant urinary protein is a challenge for precise and deep proteomic analysis of the urinary vesicle contents since it can interfere with the detection of low abundance proteins [3]. Currently, the widely used method to isolate vesicles
relies on differential centrifugations, which enrich vesicles while resulting in high
amount of THP, even in presence of reducing agents [6]. Alternative methods have been proposed to enrich UEV without massive THP interference using e.g. sucrose cushion and/or gradient isolation procedures. All of them are time consuming and labor intensive with This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 4
Proteomics - Clinical Applications
multiple centrifugation steps, which are not suitable for a practical clinical analysis. The other UEV isolation approaches reported before, including filtration, immunoaffinity, or using vesicle precipitation reagents (ExoQuick-TC) also have their distinct limitations and not widely applied [4]. Here we introduce a new approach, named hydrostatic dialysis (HD), to enrich vesicles which can easily process large amounts of urine without involving an ultracentrifugation step, thus making it more convenient and applicable for clinical use. Like traditional methods, our approach is similarly limited by remaining abundance of THP unless further processed. Thus, the main objective of our study was to decrease the THP interference by introducing trypsin digestion, while preserving the intraluminal proteome and peptidome. In our method the first morning void urine samples were collected from healthy volunteers (3 males and 2 females, age 29-43 years). No history of kidney or other general disease of any of the individuals, and no medications or alcohol was taken 12 hours prior to urine collection. No protease inhibitors or other urinary preservatives were added to the urine samples. Informed consent was obtained from all the volunteers before sample donation. The protocol was approved by the ethical committee of Dublin City University. Figure 1 shows the workflow for UEV enrichment and characterization. Briefly, 4 liters of crude urine was centrifuged at 2000g for 30 min at room temperature (RT) [7]. Supernatant (SN) was poured into a funnel connected to the 1,000kDa molecular weight cut-off (MWCO) membrane. Soluble proteins below the MWCO, yellow pigments and salts could be effectively removed from urine within around 6 hours. After washing with 200ml milliQ water, the remaining 6-8ml solution retained inside the dialysis tube (hydrostatic This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 5
Proteomics - Clinical Applications
dialysis above 1000kDa, HDa) was collected. The procedure for processing 0.5 l of urine with one HD system takes about 8 hours. Negative transmission electron microscopy (TEM) analysis was then performed to verify the presence of UEV. To eliminate the contamination of THP, HDa solution with 1mg protein (as determined by Bradford protein assay) was reduced by 10mM TRIS (carboxyethyl) phosphine (TCEP), 6M Urea, 0.1mM Ethyldiamminotetracetic acid (EDTA) and 0.2M Tris buffer pH8.8 in dark for 2 hours at RT, followed by alkylation with 20mM iodoacetamide (IAA) in dark for 2 hours. After that, 40mM N-acetyl cysteine (NAC) was added to the samples to quench the excess of IAA. Then the sample was ultrafiltrated with Vivaspin 15R ultrafiltration device having 10kDa MWCO membranes (Sartorius Stedim Biotech GmbH, Goettingen, Germany) to remove salts (10kDa), present in the HDa fraction and not belonging to UEV, and to reduce the remaining THP as much as possible. Then the sample was ultrafiltered by 30kDa MWCO Vivaspin 15R device to remove peptides. This step was repeated 3 times. The last retained fraction above 30kDa was incubated with 1% (w/v) sodium deoxycholate (DOC) to solubilize the membrane of UEV and release the intraluminal proteins which were reduced and alkylated as described above. Internal peptides were first isolated by ultrafiltration (10kDa) and then the retained solution (>10kDa) underwent three cycles of trypsin digestion with 1% DOC as previously described. The internal peptidome This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 6
Proteomics - Clinical Applications
(below 10 kDa) and internal proteome (below 30 kDa) were then acidified with 1% (v/v) formic acid (FA) to precipitate DOC and then the supernatant cleaned up by Sep-Pak C18 cartridge (Waters Associates, Milford, MA) as well [8].TEM analysis showed a heterogeneous population of nanoparticles in HDa fraction (Figure 1B). The size range (between 30 and 300 nm diameter) is consistent with that of exosomes, exosome-like and microvesicles. Each fraction during the UEV processing step of the workflow
was analyzed
by SDS-PAGE and Western blotting (Figure 1 C, D, E). After reduction, alkylation (lane 2) and digestion (lanes 3-5), the amount of intact THP (Figure 1C and E) in HDa fraction was strongly reduced. The intensive signal of Tumor susceptibility gene 101 (TSG101)(~ 46 kDa) (Figure 1 D) used as a exosome marker, was visible at the initial vesicles fractions (HDa, lane 1), suggesting the effective enrichment of UEV with our isolation method and after the trypsin digestion which did not affect the integrity of TSG101 (lane 2-5). Conversely, it disappeared after DOC treatment and trypsin digestion (lane 6). This indicates that, only after solubilising the phospholipidic bilayer by DOC, the trypsin can digest TSG101. The above results demonstrate the feasibility and efficiency of our approach, which uses trypsin digestion and ultrafiltration, to strongly reduce the interference of THP while fully preserving the integrity of (TSG101 positive) exosomes. As expected, the contaminant proteins outside UEV, particularly the THP, were cleaved into small peptides and removed by consequent 30kDa MWCO ultrafiltration. Previous studies demonstrated formidable resistance of UEV to various treatments [7], but no previous study has systematically considered the structural stability after reduction, alkylation and trypsin digestion. The Western blot results clearly indicate the structural This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 7
Proteomics - Clinical Applications
integrity of such vesicles as even 3 consecutive trypsinizations did not affect TSG101 signal. Thus, this feature also give us the opportunity to separate and purify trypsin resistant UEV, mostly exosomes, by means of simple ultrafiltration. Next, the following DOC treatment, reduction, alkylation, trypsinization, and series of ultrafiltrations could further separate proteins and peptides inside the vesicles, which gives us a reliable endoproteome and endopeptidome mapping of the UEV involved. While the urinary proteome has been widely investigated earlier in several studies, the peptidome is less well known, and it represents an important source of potential biomarkers [9]. Taking advantage of the ultrafiltration step with different MWCO filters, we isolated and characterized, for the first time, the endopeptidome of the UEVs. Internal vesicular proteome and peptidome was identified by nLC-ESI-MS/MS [10]. Lyophilized, desalted fractions were re-suspended in 50µl of 0.1% TFA and the protein concentration was estimated using the NanoDrop spectrophotometer (Thermo Scientific, Bonn, Germany). For this, 5µg of protein sample was injected into Dionex UltiMate 3000 Rapid Separation LC nano system (Thermo Scientific) coupled with an Impact HDTM mass spectrometer (Bruker Daltonics, Bremen, Germany). Peptide samples were loaded onto a Pepmap pre-column (2cm x 100µm, 5µm, Thermo Scientific), followed by separation on 50cm Nano column (0.075mm, 2µm, Acclaim PepMap100, C18, Thermo Scientific), at a flow rate of 300nl/min. Multistep 360min gradients of acetonitrile were used. The column was connected to a nanoBoosterCaptiveSprayTM (Bruker Daltonics). Mass spectrometer was operated in data-dependent-acquisition mode, using the IDAS and RT2 functionalities. Peaklists were processed using an in-house Mascot search engine (v2.4.1). Database This article is protected by copyright. All rights reserved.
www.clinical.proteomics-journal.com
Page 8
Proteomics - Clinical Applications
searching was restricted to human reviewed database Swiss-Prot (accessed Apr 2014, 544,996 sequences; 193,815,432 residues). Mass tolerances were set at 20ppm for MS and 0.05Da for MS/MS. For digested samples (proteome), trypsin and Carbamidomethyl(C) were set as enzyme and as fixed modification, respectively. Automatic decoy database search and built-in Percolator algorithm were used to rescore search results and calculate posterior error probabilities for each peptide-spectrum match. All results were filtered to achieve global FDR
