Protocols
단백질 PTM분석
  • 특정 단백질이 신호전달 과정에서 PTM에 따라 신호를 on/off하는 것 같아서 그 단백질의 PTM을 단백체 방법으로 분석하는 경우가 있다.
  • 신호를 준 조건에서 그 단백질을 IP하고 proteomic method를 활용해 관련된 PTM을 알아 낼 수 있는데, 이 경우 시료 준비과정에서 주의할 점이 있다.
  • Western blot으로 타겟 단백질을 확인하는 것만으로는 안되며, IP한 시료가 실제로 PTM분석에 적당한지 꼭 확인해봐야 한다.
공동연구
  • 단백체분석 또는 단백질 질량분석은 다양한 방식이 있다. 이 때, 단백체 분석의 목적 등을 정확히 파악해야 어떻게 분석하는게 나은지, 어떤 과정으로 시료를 전처리해야하는지 전략을 짤 수 있다.
  • 공동연구가 잘되려면 되도록 정확하고 풍부한 정보를 주고받아야 한다.
  • 첨부파일은 그런 조건을 만족하는 몇가지 실례이다.
Proteome Data
Proteome data resource page는 삼성서울병원이 보건산업진흥원으로부터 수주한 연구중심병원과제(과제코드: HI14C3484)에서 지원받아 운영하고 있습니다.
This data resource page is supported by the research hospital program that Samsung Seoul Hospital has received (HI14C3484) from the Korea Health Industry Development Institute.
  1. cRFP (common Repository of FBS Proteins)
  2. Human Wharton's Jelly MSC Secretome (serum effect)
  3. U87MG Secretome (serum effect)
  4. Lung Cancer Cell Secretomes
  5. Human Protein N-termini
  6. Breast Cancer Cell Secretomes
  7. FBS proteins frequently found in secretome as contaminants
cRFP (common Repository of FBS Proteins)
Title
  • cRFP (common Repository of FBS Proteins)
Description
  • Mass spectrometric (MS) data of human cell secretome are usually searched against conventional human database. However, such procedure may cause a number of false identifications due to contamination of the secretome with fetal bovine serum (FBS) proteins.
  • We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretome.
  • cRFP is a small supplementary sequence list of high abundant fetal bovine serum proteins added to the reference database in use.
  • The list was constructed by MS analysis of FBS proteins directly.
  • The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins.
  • The result will be more reliable with less false-positive and false-negative identifications than using the conventional database only.
  • It can be generally applied to cell secretome of serum-free medium, extracellular vesicle proteome, etc.
How to use:
  1. Download the cRFP FASTA file below.
  2. Designate the cRFP file as a contaminant DB in your analysis platform such as Proteome Discoverer.
    Otherwise, merge the file with your database in use and then use the merged one for your search DB.
Downloadable files:
Sample processing protocol
Data processing protocol
Remarks
  • Mar 30, 2015: Publication of the concept and an experimental evidence using secretome of cell-free medium
  • Jul 1, 2019: First release of cRFP fasta file (199 entries)
  • Sep 10, 2019: More evidences for the general use of cRFP. Published (Epub) in Jorunal of Proteome Research as a technical note: Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome
  • May 1, 2020: Second release of cRFP (274 entries)
    • Mass spec: LTQ-Orbitrap LC-MS
    • Prefractionation: 12 high pH reversed-phase fractions of FBS
    • Search: with Uniprot reference proteome database (release 200227); 23,845 entries
    • Filtering: unique peptide > 1
    • cRFP2 = cRFP + newly identified 208 proteins
      Entry DescriptionProtein count
      Only in cRFP Identified previously but not at cRFP246
      Overlap 62
      Sequence updated Sequences are updated according to the new protein DB38
      Newly identified Proteins identified at CRFP2 only128
      Sum 274
Date
  • July 1, 2019
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Human Wharton's Jelly MSC Secretome (serum effect)
Title
  • Human Wharton's Jelly MSC proteins differentially secreted in serum-containing medium compared to serum free medium
Description
  • Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the majority of mass spectrometric cell secretome studies have been performed using serum-free medium (SFM). On the other hand, serum-containing medium (SCM) is not recommended very much because the secretome obtained with SCM is easily contaminated with fetal bovine serum (FBS) proteins.
  • Through the combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed-SILAC (pSILAC), we analyzed differentially secreted proteins between SFM and SCM in a mesenchymal stem cell derived from human Wharton’s jelly (hWJ-MSC).
  • In most cases, the bioinformatic tools predicted a protein to be truly secretory when the secretion level of the protein was more in SCM than in SFM.
  • hWJ-MSC proteins secreted more in SCM included several positive markers of MSC paracrine factors implicated in angiogenesis, neurogenesis and osteogenesis, and upstream regulators of cell proliferation.
Sample processing protocol
  • hWJ-MSC was cultured in DMEM (Gibco, Rockville, MD) supplemented with 10% FBS (Gibco, Rockville, MD), 1% penicillin and streptomycin (Gibco, Rockville, MD) at 37 °C in a humidified 95% air, 5% CO2 incubator. Cells were seeded 2.2E6 cells in 100-mm culture dishes (Nunc, Naperville, IL) and incubated for 24 h.
  • For BONCAT-pSILAC, the cells were depleted of methionine, lysine, and arginine in a depletion medium (DMEM non-GMP formulation without methionine, arginine, and lysine; Gibco) with 10% dialyzed FBS for 1 h, and then incubated for 24 h in the same medium supplemented with 1 mM AHA and either 0.398 mM [13C6,15N4]L-arginine and 0.789 mM [13C6,15N2]L-lysine (Cambridge Isotope Laboratories, Inc.) as heavy-isotope with 10% dialyzed FBS or 0.398 mM [13C6]L-arginine and 0.789 mM [4,4,5,5-D4]L-lysine (Cambridge Isotope Laboratories, Inc.) as medium-isotope without FBS.
  • After incubation, culture media were carefully collected. Floating cells and cellular debris were removed by centrifugation (400 × g, 10 min, 4 °C), followed by sterile filtration (pore size: 0.22 μm, Millipore, MA).
  • Newly synthesized and secreted proteins were enriched from concentrated media using the Click-iT® Protein Enrichment Kit (Invitrogen C10416), employing the vendor’s protocol with slight modifications. Typically, 100 µL of agarose resin slurry was used for concentrated SCM.
  • The SCM was concentrated up to ~250 µL through ultrafiltration using ‘Amicon Ultra-15’ centrifugal filter devices (Millipore, MA), and was exchanged into a denaturation buffer containing 8 M urea and 100 mM Tris (pH 8.2) by repeating dilution-ultrafiltration twice.
  • CuAAC reaction was carried out overnight at RT after mixing the sample with appropriate resin and solutions supplied by the vendor. The whole mixture adjusted to 0.5 ml with water was then transferred on to a 0.22-µm centrifugal filter unit (Millipore).
  • All the water-soluble materials were removed by spinning the filter unit, leaving only the resin.
  • After then, the resin with proteins attached was treated with 20 mM DTT in 0.5 ml of 1% SDS at 70 °C for 15 min, and then with 40 mM iodoacetamide in 0.5 ml of 1% SDS at RT for 30 min in the dark, and washed with 0.5 ml of 1% SDS, 0.5 ml of 8 M urea/100 mM Tris (pH 8.2) and 0.5 ml of 20% acetonitrile. Each washing step was repeated at least five times.
  • The washed resin was resuspended in a buffer containing 100 mM Tris (pH 8.2), 2 mM CaCl2, and 10% acetonitrile, mixed with 0.5 µg trypsin and incubated for 16 h at 37 °C.
  • The peptides of trypsin cleavage product were collected by centrifugation and the resin was washed with 0.5 mL of water. The two solutions were combined, and acidified with 0.5% TFA.
  • The samples were analyzed by LC-MS/MS using Q Exactive mass spectrometer (Thermo Fisher Scientific). One microgram of sample reconstituted in 0.4% acetic acid was injected into a reversed-phase C18 column (20 cm × 75 μm i.d., 3 μm, 120 Å, packed in-house; Dr. Maisch GmbH) on an Eksigent nanoLC-ultra 1D plus system at 95% solvent A and 5% solvent B. The peptides were eluted with a linear gradient from 5% to 40% solvent B over 200 min followed by 80% solvent B wash and 95% solvent A re-equilibration at a flow rate of 300 nL/min with a total run time of 230 min.
  • Survey full-scan MS spectra (m/z 350–1800) were acquired at a resolution of 70000. Source ionization parameters were as follows: spray voltage, 2.5 kV; capillary temperature, 300 °C; and s-lens level, 44.0. The MS peak width at half height was <30 s.
  • The MS/MS spectra of the 12 most intense ions from the MS1 scan with a charge state ≥2 were acquired with the following options: resolution, 17500; isolation width, 2.0 m/z; normalized collision energy, 27%; ion selection threshold, 4.00E ± 03 counts; and peptide match, ‘preferred’.
Data processing protocol
  • Proteome Discoverer 2.2.0.388 was used with three search engines: Sequest HT, Mascot and MS Amanda. Each search engine was set to medium- and heavy-isotope of SILAC as variable modifications with precursor mass tolerance up to 15 ppm and an allowed fragment mass deviation of 0.05 Da. Search parameters also included two missed trypsin cleavage sites, cysteine carbamidomethylation as fixed modification, methionine oxidation and N-terminal protein acetylation as variable modifications.
  • Human UniProtKB reference proteome database (released in January 2016; 20,218 entries) combined with a list of experimentally validated FBS proteins (199 entries) was used.
  • Peptide and protein results were filtered to 1% FDR.
  • Only the proteins with at least one unique peptide supported by all three search engines were accepted into the final result list.
  • The H/M ratios of peptides were calculated by dividing the intensities of heavy-isotope by the medium-isotope intensities and then transformed to log2 values.
  • For missing value imputation, the smallest integer greater than the largest log2 peptide ratio was given: a value of −8 was given to the peptides whose heavy isotope was not observed; a value of 8 was given to the peptides whose medium isotope was not observed. Protein ratio was the geometric mean of all unique peptide ratios.
Remarks
  • Jihye Shin, Jiheon Rhim, Yumi Kwon, Sun Young Choi, Sungho Shin, Chul-Won Ha, Cheolju Lee (2019 Feb 28) Comparative analysis of differentially secreted proteins in serum-free and serum-containing media by using BONCAT and pulsed SILAC. Sci Rep 9 (1): 3096. doi: 10.1038/s41598-019-39650-z.
Date
  • 28 February 2019
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U87MG Secretome (serum effect)
Title
  • U87MG proteins differentially secreted in serum-containing growth medium compared to serum free medium
Description
  • Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the majority of mass spectrometric cell secretome studies have been performed using serum-free medium (SFM). On the other hand, serum-containing medium (SCM) is not recommended very much because the secretome obtained with SCM is easily contaminated with fetal bovine serum (FBS) proteins.
  • Through the combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed-SILAC (pSILAC), we analyzed differentially secreted proteins between SFM and SCM in a cancer-derived human cell, U87MG.
  • In most cases, the bioinformatic tools predicted a protein to be truly secretory when the secretion level of the protein was more in SCM than in SFM.
Sample processing protocol
  • U87MG was cultured in DMEM (Gibco, Rockville, MD) supplemented with 10% FBS (Gibco, Rockville, MD), 1% penicillin and streptomycin (Gibco, Rockville, MD) at 37 °C in a humidified 95% air, 5% CO2 incubator.
  • All sample processing procedures were the same as for hWJ-MSC.
Data processing protocol
  • All data handling procedures were the same as for hWJ-MSC.
Remarks
  • Jihye Shin, Jiheon Rhim, Yumi Kwon, Sun Young Choi, Sungho Shin, Chul-Won Ha, Cheolju Lee (2019 Feb 28) Comparative analysis of differentially secreted proteins in serum-free and serum-containing media by using BONCAT and pulsed SILAC. Sci Rep 9 (1): 3096. doi: 10.1038/s41598-019-39650-z.
Date
  • 28 February 2019
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Lung Cancer Cell Secretomes
Title
  • LC-MS/MS analysis of serum-free medium secretomes from six lung cancer cell lines (A549, Calu-1, H1299, H23, H460, and H520)
Description
  • 2,992 non-redundant human proteins were identified from the six cell lines, among which 942, 1498, 1114, 1390, 1328, and 1128 proteins were from A549, Calu-1, H1299, H23, H460, and H520, respectively.
  • As a negative control, α-tubulin was examined in cell secretomes by Western blot analysis. The cytoskeleton protein was clearly detected in cell extracts but not in conditioned media, indicating that our secretome was rarely contaminated with intracellular proteins released from ruptured cells.
Sample processing protocol
  • The lung cancer cell lines A549, Calu-1, H1299, H23, H460 and H520 were cultured in RPMI 1640 (Gibco, Rockville, MD, USA) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C in a humidified 95% air, 5% CO2 incubator.
  • Cells were grown to approximately 70% confluence and rinsed carefully three times with serum-free medium (SFM) at room temperature.
  • The cells were incubated in SFM at 37°C for 12 h to minimize the release of cytosolic proteins into the surrounding medium due to cell death.
  • After incubation, the conditioned media were carefully collected and 2 mM PMSF and 1 mM EDTA were added as protease inhibitors.
  • Floating cells and cellular debris were removed by centrifugation (400 × g, 10 min, 4°C), followed by sterile filtration (pore size: 0.22 μm, Millipore, MA, USA).
  • The media were concentrated and exchanged into buffer consisting of 8 M urea, 75 mM NaCl, and 50 mM Tris (pH 8.2) by ultrafiltration using Amicon Ultra-15 centrifugal filter devices (Millipore).
Data processing protocol
  • In-solution digestion with trypsin
  • Reversed-phase Magic C18aq column (15 cm × 75 μm, 200Å, 5U).
  • Flow rate of 0.4 μL/min across the analytical column with a linear gradient of 5–40% acetonitrile in 0.1% formic acid over 90 min.
  • LTQ-XL mass spectrometer (Thermo Scientific, San Jose, CA)
  • MS/MS spectra were searched using SEQUEST (TurboSequest version 27, revision 12) against UniProtKB database (released in March 2012) plus cRFP, the experimentally validated 199 FBS proteins.
  • Two trypsin-missed cleavages, peptide mass tolerances of ± 0.5 and ± 2 Da for MS/MS and MS, fixed modification of carbamidomethylation at cysteine (+ 57.02 Da), and variable modification of oxidation at methionine (+ 15.99 Da).
  • Peptide assignment and validation were performed using the Trans-Proteomic Pipeline (TPP, version 4.0, http://www.proteomecenter.org).
  • Percolator was used to calculate q-values as measures of statistical confidence for each PSM. We filtered the output with q < 0.01.
Remarks
  • Jihye Shin, Sang-Yun Song, Hee-Sung Ahn, Byung Chull An, Yoo-Duk Choi, Eun Gyeong Yang, Kook-Joo Na, Seung-Taek Lee, Jae-Il Park, Seon-Young Kim, Cheolju Lee, Seung-Won Lee (2017 Aug 24) Integrative analysis for the discovery of lung cancer serological markers and validation by MRM-MS. PLoS One 12 (8): e0183896. doi: 10.1371/journal.pone.0183896.
Date
  • August 24, 2017
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Human Protein N-termini
Title
  • Comprehensive analysis of human protein N-termini
Description
  • Various forms of protein (proteoforms) are generated by genetic variations, alternative splicing, alternative translation initiation, co- or post-translational modification and proteolysis. Different proteoforms are in part discovered by characterizing their N-terminal sequences.
  • We introduce an N-terminal-peptide-enrichment method, Nrich. Filter-aided negative selection formed the basis for the use of two N-blocking reagents and two endoproteases in this method.
  • We identified 6,525 acetylated (or partially acetylated) and 6,570 free protein N-termini arising from 5,727 proteins in HEK293T human cells.
Sample processing protocol
  • Nrich consisted of three major experimental steps:
    1. The first step was to distinguish between endogenous Nα -acetylated and endogenous free N-termini. This was done by blocking α and ε primary amines of proteins with propionic anhydride or D6-acetic anhydride.
    2. Amine-blocked proteins were digested with trypsin or GluC-endoprotease using FASP methods for N-blocking reagent removal and buffer exchange.
    3. Newly-generated internal peptides containing free α-amine were removed with a N-hydroxysuccinimide (NHS)-activated agarose resin.
  • The detailed sample processing procedure can be found in the paper below.
Data processing protocol
  • The detailed data processing protocol can be found in the paper below.
Remarks
  • Jeonghun Yeom, Shinyeong Ju, YunJin Choi, Eunok Paek, Cheolju Lee (2017 Jul 26) Comprehensive analysis of human protein N-termini enables assessment of various protein forms. Sci Rep 7 (1): 6599. doi: 10.1038/s41598-017-06314-9.
Date
  • 26 July 2017
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Breast Cancer Cell Secretomes
Title
  • Secretomes of 4 breast cancer cell lines (MCF‐7, MDA‐MB‐231, SK‐BR‐3, and Hs578T) from serum free conditioned medium
Description
  • The secretomes of four breast cancer cell lines (Hs578T, MCF‐7, MDA‐MB‐231, and SK‐BR‐3) were profiled with liquid chromatography–tandem mass spectrometry analysis.
  • A total of 1410 proteins were identified with less than 1% false discovery rate - 936, 603, 585, and 475 human proteins, respectively.
Sample processing protocol
  • Hs578T, MCF‐7, MDA‐MB‐231, and SK‐BR3 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco, Rockville, MD, USA) at 37°C with 5% CO2 incubator. In the case of Hs578T, 0.01 mg/mL insulin (Sigma‐Aldrich, St. Louis, MO, USA) was further supplemented.
  • Cells were grown to ∼70% confluency (∼1.6E7 cells) in 150 mm culture dishes.
  • The cell monolayer was rinsed carefully with serum-free medium (SFM) three times at RT. Then, the cells were incubated in the SFM at 37 °C for about 12 h.
  • After incubation, the SFM was carefully collected; 2 mM PMSF and 1 mM EDTA were added as protease inhibitors.
  • Floating cells and cellular debris were removed by centrifugation (400g, 10 min, 4 °C), followed by sterile filtration (pore size: 0.22 μm, Millipore, MA). The conditioned medium was concentrated through ultrafiltration using “Amicon Ultra-15” centrifugal filter devices (Millipore, MA).
  • Secreted proteins were precipitated by using acetone at −20 °C for 1 h and then dissolved in buffer consisting of 8 M urea, 75 mM NaCl, 50 mM Tris (pH 8.2).
  • In-solution digestion was performed on the secretome sample.
  • Tryptic digests were separated using a reversed phase Magic C18 column (75 μm) on an Agilent 1200 HPLC system, (Agilent Technologies, Santa Clara, CA) with a linear gradient of 10–40% acetonitrile containing 0.1% formic acid for 90 min (400 nL/min). The HPLC system was coupled to an LTQ‐XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA).
Data processing protocol
  • All tandem mass spectral data were searched by using the SEQUEST algorithm (TurboSequest version 27, revision 12) against the human UniProtKB database (released in March 2012) supplemented with 199 experimentally validated FBS contaminant sequences (cRFP).
  • Two trypsin missed cleavages, fixed modification of carbamidomethylation at cysteine (+57.02 Da), and variable modification of oxidation at methionine (+15.99 Da) were allowed. Mass tolerances for MS/MS and MS were set to ±0.5 and ±2 Da, respectively.
  • Peptide and protein assignment and validation (false discovery rate <1%) were carried out using the Trans Proteomic Pipeline version 4.5
Remarks
  • Jihye Shin, Gamin Kim, Jong Won Lee, Ji Eun Lee, Yoo Seok Kim, Jong-Han Yu, Seung-Taek Lee, Sei Hyun Ahn, Hoguen Kim, Cheolju Lee (2016 Jun) Identification of ganglioside GM2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes. Cancer Sci 107 (6): 828-835. doi: 10.1111/cas.12935.
Date
  • March 22, 2016
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FBS proteins frequently found in secretome as contaminants
Title
  • FBS proteins frequently found in secretome as contaminants
Description
  • Mass spectrometric (MS) data of human cell secretome are usually searched against conventional human database. However, such procedure may cause a number of false identifications due to contamination of the secretome with fetal bovine serum (FBS) proteins. FBS proteins are very hard to completely remove from the cells. Therefore, we analyzed FBS proteins as a whole and the FBS proteins that were attached to the cell culture plate in order to identify FBS proteins frequently found in secretome as contaminants.
Sample processing protocol
  • In solution digestion with trypsin of the following FBS samples collected in three different ways:
    1. Cell-free polystyrene-plates (150 mm) containing RPMI1640 and DMEM media supplemented with 10% FBS were incubated for 2 days. The plates were washed with serum-free medium (SFM) three times and incubated for 12 h. The media were collected and concentrated 250 times (50 mL to 200 μl) by ultrafiltration.
    2. To each of the empty plates, 400 μL of lysis buffer was added and the surfaces of the plates were physically scraped. The lysis buffers from the cell-free conditioned media were collected and analyzed separately.
    3. Thirdly, commercial FBS was directly used for proteome analysis.
  • LC-MS/MS on a reversed-phase Magic C18aq column (15 cm × 75 μm, 200Ǻ)
  • 5–40% acetonitrile gradient in 0.1% formic acid.
  • LTQ-XL mass spectrometer (Thermo Scientific, San Jose, CA).
Data processing protocol
  • SEQUEST in Proteome Discoverer 1.4 (Thermo Fisher Scientific, version 1.4.0.288).
  • Percolator was used to calculate q value as a statistical confidence measure for each PSM.
Remarks
  • All MS files are available from the PeptideAtlas database (dataset identifier: PASS00579)
  • Jihye Shin, Gamin Kim, Mohammad Humayun Kabir, Seong Jun Park, Seoung Taek Lee, Cheolju Lee (2015 Mar 30) Use of Composite Protein Database including Search Result Sequences for Mass Spectrometric Analysis of Cell Secretome. PLoS One 10 (3): e0121692. doi: 10.1371/journal.pone.0121692
Date
  • Mar 30, 2015
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Data Code
Title
Description
Sample processing protocol
Data processing protocol
Remarks
Date
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