Deep Phosphotyrosine Proteomics by Optimization of

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Deep phosphotyrosine proteomics by optimization of phosphotyrosine enrichment and MS/MS parameters Yuichi Abe, Maiko Nagano, Asa Tada, Jun Adachi, and Takeshi Tomonaga J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00576 • Publication Date (Web): 28 Nov 2016 Downloaded from http://pubs.acs.org on November 29, 2016

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Journal of Proteome Research

Deep phosphotyrosine proteomics by optimization of phosphotyrosine enrichment and MS/MS parameters

AUTHOR NAMES Yuichi Abe1, Maiko Nagano1, Asa Tada1, Jun Adachi1*, Takeshi Tomonaga1*

AUTHOR ADDRESS 1

Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and

Nutrition, Ibaraki, Osaka 567-0085, Japan

KEYWORDS Phosphotyrosine, Immunoprecipitation, Phosphorylation signaling, Caner, Kinome

ACS Paragon Plus Environment

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ABSTRACT

Phosphorylation is a major post-translational modification that regulates protein function, with phosphotyrosine (pY) modifications being implicated in oncogenesis. However, global profiling of pY statuses without treatment with a tyrosine phosphatase inhibitor such as pervanadate is still challenging due to the low occupancy of pY sites. In this study, we greatly improved the identification of pY sites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by optimization of both the pY-immunoprecipitation (pY-IP) protocol and the LC-MS/MS parameters. Our highly sensitive method reproducibly identified more than 1000 pY sites from 8 mg of protein lysate without the need for tyrosine phosphatase inhibitor treatment. Furthermore, more than 30% of the identified pY sites were not assigned in the PhosphositePlus database. We further applied our method to the comparison of pY status between PC3 cells with and without treatment using the epidermal growth factor receptor (EGFR) inhibitor Erlotinib. Under Erlotinib treatment, we observed not only a decrease in well-known modes of EGFR downstream signaling but also modulations of kinases that are not relevant to the EGFR cascade, such as PTK6 and MAPK13. Our newly developed method for pY proteomics has the potential to reveal unknown pY signaling modes and to identify novel kinase anti-cancer targets.


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Introduction Protein phosphorylation at serine, threonine, and tyrosine residues is important for the regulation of protein functions such as enzymatic activity. Many studies have shown that abnormality in the phosphotyrosine (pY) signaling pathway is closely linked to oncogenesis and other diseases 1. Therefore, accurate measurements of global pY statuses could contribute to the general understanding of pY signaling networks and identifying therapeutic targets for various diseases 2-5

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Phosphoproteomic analysis using mass spectrometry is a powerful tool for analyzing global protein phosphorylation. In global phosphoproteomics, phosphopeptides are usually enriched using techniques such as immobilized metal affinity chromatography (IMAC) 6, metal oxide affinity chromatography (MOC) 7, and hydroxyl acid-modified metal oxide chromatography (HAMMOC) 8. However, significant analytical difficulty is encountered in pY proteomics because pY modifications constitute only a small fraction of the exhibited phosphorylation modifications. A previous study reported that the ratio of pY in HeLa cells is less than 2% 9. To overcome this problem, further enrichment of pY peptides by immunoprecipitation with pY antibodies has been explored 10-12. However, global pY identification remains problematic, especially without the use of tyrosine phosphatase inhibitors. In this study, we developed an improved pY enrichment protocol and an optimized liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for identifying pY peptides in samples. Furthermore, we applied our newly developed method to the measurement of the global pY status of PC3 lung adenocarcinoma cells with and without treatment of the kinase inhibitor drug Erlotinib.


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Experimental Section Reagents and antibodies Dulbecco's modified Eagle's medium (DMEM), Iscove’s modified Dulbecco’s medium (IMDM), and Chemi-Lumi Super were purchased from Nacalai Tesque (Kyoto, Japan). Magnetic Dynabeads Protein G for immunoprecipitation, Protein G Agarose beads, a tandem mass tag (TMT) 10-plex isobaric label reagent set, SimplyBlue SafeStain, fetal bovine serum (FBS), and Penicillin-Streptomycin were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Oasis HLB cartridges were obtained from Waters (Milford, MA, USA). Erlotinib was purchased from Selleck (Houston, TX, USA). Trypsin, PhosSTOP phosphatase inhibitor cocktail, and cOmplete protease inhibitor cocktail were obtained from Roche (Basel, Switzerland). Phosphate-buffered saline (PBS) and Tris-buffered saline (TBS) tablets were obtained from Takara (Shiga, Japan). A detergent-compatible (DC) protein assay kit was purchased from Bio-Rad (Hercules, CA, USA). XV Pantera gel (5–20%) was obtained from DRC (Tokyo, Japan). An ImmunoStar LD kit was obtained from Wako (Osaka, Japan). Cell Counting Kit-8 was purchased from Dojindo (Kumamoto, Japan). The Prism 6 software package was obtained from GraphPad Software (La Jolla, CA, USA). Phospho-Tyrosine (P-Tyr-1000) MultiMab, pERK1/2 T202/Y204 (D13.14.4E), pMEK1/2 S217/221 (41G9), and pMAPK13 T180/Y182 (D3F9) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). ERK1/2 (MK1) and MEK1/2 (9G3) antibodies were obtained from SantaCruz (Dallas, TX, USA). PTK6 (2H12B8) antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA), MAPK13 antibody was purchased from Calbiochem (San Diego, CA, USA), pPTK6 Y447 antibody was purchased from LifeSpan


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Biosciences (Seattle, WA, USA), and GAPDH (6C5) antibody was purchased from Abcam (Cambridge, UK).

Cell culture and sample preparation HCT116 and PC3 cells were cultured in DMEM and IMDM, respectively, at 37 °C under 5% CO2. Both media were supplemented with 10% FBS and 1% Penicillin-Streptomycin. The cells were harvested after washing with ice-cold PBS buffer containing PhosSTOP and cOmplete. Collected cells were pelleted using centrifugation and flash-frozen with liquid nitrogen. Frozen cell pellets were stored at -80 °C prior to use.

Cell lysis, cleavage with trypsin, depletion of surfactant, and primary Fe3+ IMAC The cells were lysed in phase transfer surfactant (PTS) buffer (50 mM ammonium bicarbonate, 12 mM sodium deoxycholate, 12 mM sodium lauroyl sarcosinate) supplemented with PhosSTOP and cOmplete protease inhibitors. The protein concentration was determined using a DC protein assay according to the manufacturer’s protocol. A 2.0 or 8.0 mg protein lysate sample was reduced, alkylated, and subsequently trypsinized as described previously 13. The surfactant was removed as described previously 14. Primary enrichment of phosphopeptides from whole peptides was performed with Fe3+ IMAC resin as described previously 15.


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TMT labeling In the experiments using PC3 cells with and without treatment with Erlotinib, phosphopeptides were labeled with the TMT 10-plex reagent before pY-immunoprecipitation (pY-IP). For each label, phosphopeptides enriched from 0.8 mg of protein digest were mixed with the TMT labeling reagent according to the manufacturer’s protocol. Labeled phosphopeptides were then mixed and dried. The mixture was used for the following pY-IP experiments. The quantitation data for the 127C label was discarded because its pY intensity was unusually low compared with the other samples (Figure S-1).

IP with pY antibody and secondary Fe3+ IMAC For the IP with pY antibody, 12.5 or 50 µg of pY1000 antibody was conjugated to magnetic Dynabeads Protein G or Protein G Agarose beads at room temperature for 1 h. Phosphopeptides enriched from 2.0 or 8.0 mg of protein digest were re-suspended in 50 or 150 µL, respectively, of IP buffer (5 mM tris-HCl (pH 7.5), 5 mM NaCl). The dissolved phosphopeptides were mixed with antibody-conjugated Dynabeads or Agarose beads and stirred at 4 °C for 20 h. In the case of pY-IP with pre-IMAC peptides, 8.0 mg of the digested peptides was directly re-suspended in 150 µl of IP buffer and then mixed with antibody-conjugated Dynabeads. Then, the supernatant was removed from the Dynabeads using a magnetic stand. When using agarose beads, the supernatant was removed after centrifugation at 2,500 g for 3 min. The Dynabeads or Agarose beads with pY peptides were then washed four times with IP buffer. pY peptides on the Dynabeads or Agarose


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beads were eluted two times with 50 µl of 60% acetonitrile (ACN)/0.1% TFA. The time taken for the elution of the pY peptides was 3 and 1 min for the first and second elutions, respectively. For depletion of pY1000 antibodies (i.e., secondary Fe3+ IMAC), the eluted pY peptides were applied to Fe3+ IMAC columns on a C18 disc in a stop-and-go extraction tip (StageTip) 16. After being loaded onto the Fe3+ IMAC resin, the pY peptides were washed three times with 60% ACN/0.1% TFA and one time with 0.1% TFA. Then, the pY peptides were transferred to a C18 disc with 100 µl of 1% H3PO4 and 100 µl of 500 mM K2HPO4. After transfer of pY peptides from Fe3+ IMAC to the C18 disc, the C18 disc was desalted with 0.1% TFA and pY peptides were eluted with 60% ACN/0.1% TFA. After drying the elutes by speed-vac, the pY peptides were re-suspended in 10 µl of 2% ACN/1% TFA for LC-MS/MS analysis.

Mass spectrometry analysis The liquid chromatography and mass spectrometry parameters were modified from those of a previous study 13. The nano-LC gradient was performed at 280 nL/min and consisted of a linear gradient of buffer B from 5 to 30% B over 45 min. The Q Exactive instrument was operated in data-dependent mode. Dynamic exclusion was set to 10 s. The 4, 6, or 12 most intense multiply charged ions (z ≥ 2) were sequentially accumulated to a 2 × 105 target value and fragmented in an octopole collision cell by higher-energy collisional dissociation with a maximum injection time of 120, 240, or 360 ms and a resolution of 35,000.


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For pY proteomics using TMT labeling, we analyzed using a Q Exactive Plus unit. The LC parameters were modified from those of a previous study 13. The nano-LC gradient corresponded to the parameters used with the Q Exactive method. The experiments with the Q Exactive Plus unit were operated in data-dependent mode. Survey full-scan MS spectra (m/z 350−1800) were acquired in the Orbitrap with a resolution of 70,000 after accumulation of ions to a 1 × 106 target value. Dynamic exclusion was set to 10 s. The six most intense multiply charged ions (z ≥ 2) were sequentially accumulated to a 1 × 105 target value and fragmented in the octopole collision cell by higher-energy collisional dissociation with a maximum injection time of 240 ms and a resolution of 35,000. Typical mass spectrometric conditions were as follows: spray voltage, 2 kV; no sheath and auxiliary gas flow; heated capillary temperature, 250 °C; normalized HCD collision energy, 30%. The MS/MS ion selection threshold was set to 1.3 × 104 counts. A 1.0 Da isolation width was chosen.

Data processing & analysis Raw data were processed using MaxQuant 1.5.1.2 supported by the Andromeda search engine for peptide identification 17. Search of MS/MS spectra against the UniProt human database (release 2011_11) combined with 262 common contaminants was performed using the Andromeda search engine. Enzyme specificity was set to a C-terminal of Arg or Lys with the allowed cleavage at proline bonds and a maximum of two missed cleavages. Carbamidomethylation of cysteine residues, TMT tags on lysine residues, and peptide N termini were set as a fixed modification, and methionine oxidation and/or serine, threonine, and tyrosine phosphorylation were set as variable modifications. Peptides and proteins were accepted with a


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Deep Phosphotyrosine Proteomics by Optimization of

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