* Skyline LC SRM. 2 Skyline LC-MS/MS. Skyline University of Washington Windows.

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1 Proteome Letters * * fmatsuda@ist.osaka-u.ac.jp LC SRM LC SRM 1 LC-MS/MS LC 1 3 SRM SRM LC-MS Skyline SRM LC-MS LC SRM 2 Skyline Skyline University of Washington Windows 4,5 SRM SRM Skyline Skyline Japanese Proteomics Society

2 Proteome Letters Table µg/ml 2 15 ml 3000 rpm, 5 min 3 50 mm Hepes ph mm Dithiothreitol DDT 100 mm KCl 5 mm EDTA Complete protease inhibitors cocktail 1 1 ml ml 6 mm mm PCR min 2.5 min rpm, 5 min ml 8 Bio-RAD RC DC protein assay Bio-Rad, Reinach, Switzerland 3 Proteomics Sample Preparation Kit for Cultured Cell, #FMR Table 1 8 StageTip 9,10 4 LC- LC- Table 2 MRMplus Retention Time Marker FMR SRM 11 LC-MS LC nanoviper fingertight fitting 12 LC Table 2 LC- LC-20ADnano A B C nl/min min [B%] 0 [0%] 7 [0%] 45 [35%] 50 [100%] 65 [100%] 67 [75%] 75 [0%] 5 40 microl/min L-column ODS 2 3 microm, 0.1*150 mm, CERI Japan L-column Fortis tip (AMR) nano-spray interface (AMR, Japan) LCMS L/min DL 150 C 200 C Q1 Low Q3 Low CID 310 kpa kv 2 ESI AMR Fortis tip AMR 1 1 Fig. 1 LC- Q-Tof SWATH 13

3 Proteome Letters Fig. 1 Long term stability of nanolc-ms/ms analyses SRM chromatograms of standard peptides (FMR-002, Funakoshi, Japan) were obtained at (a) 3/2/2015, (b) 18/2/2015, and (c) 3/3/2015 during a large scale targeted proteome analyses. LC LC 1 8 LC- 5 SRM SRM 3,8 SRM SRM Uchida Table SRM 3.55 SRM SRM [M+2H] 2+ y 81.9 [M+2H] 3+ y SRM m/z y 1 m/z SRM Gnd SRM Gnd FASTA Skyline 3 SRM LC-MS LCMS Gnd 457 SRM 4 SRM 5 SRM SRM SRM TSQ Vantage 15 Skyline

4 Proteome Letters LCMS-8040 Q1 CE=0.03 [Q1 m/z]+4.0 SRM MCF-7 Fig. 2 SRM SRM Table SRM SRM N N 5 LC SSRCalc K R KK, RR, KP RP 16 4 KR, RK 9 Peptide Atlas [M+2H] 2+ [M+3H] 3+ y b [M+2H] 2+ y [M+2H] 2+ b [M+3H] 3+ y [M+3H] 3+ b [M+2H] 2+ y Fig. 2 Targeted proteome analysis using a converted SRM assay method Targeted proteome data of the central metabolism related enzymes were successfully acquired from MCF-7 cells using a SRM assay method for LCMS8040 (Shimadzu Co.) converted from that of TSQ Vantage (Thermo Scientific).

5 Proteome Letters SILAC Stable Isotope Labeling using Amino acids in Cell culture 13 C LC-MS SILAC [U- 13 C] SRM [U- 13 C] S288C 13 C Skyline 13 C SRM SRM LC-MS Skyline C Skyline LC-MS NEDO ;84: Schiess R, Wollscheid B, Aebersold R. Targeted proteomic strategy for clinical biomarker discovery. Mol Oncology. 2009;3: Marx T. Targeted proteomics. Nature Methods. 2013;10: MacLean B, Tomazela DM, Shulman N, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26: Skyline edu/labkey/project/home/software/skyline/begin.view 6 pdf 7 Picotti P, Bodenmiller B, Mueller LN, et al. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell. 2009;138: Uchida Y, Tachikawa M, Obuchi W, et al. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/ MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood-brain barrier in ddy, FVB, and C57BL/6J mice. Fluids Barriers CNS. 2013;10:21. 9Stage Tip2008;57: ,

6 Proteome Letters Rappsilber J, Mann M, Ishihama Y. Protocol for micropurification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc. 2007;2: pdf 12 reagents-accessories/nano-viper-fingertight/lp html 13 Gillet LC, Navarro P, Tate S, et al. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteomics. 2012;11:O Costenoble R, Picotti P, Reiter L, et al. Comprehensive quantitative analysis of central carbon and amino-acid metabolism in Saccharomyces cerevisiae under multiple conditions by targeted proteomics. Mol Syst Biol. 2011;7: Drabovich AP, Pavlou MP, Dimitromanolakis A, Diamandis EP. Quantitative analysis of energy metabolic pathways in MCF-7 breast cancer cells by selected reaction monitoring assay. Mol Cell Proteomics. 2012;11: Matsuda F, Ogura T, Tomita A, et al. Nano-scale liquid chromatography coupled to tandem mass spectrometry using the multiple reaction monitoring mode based quantitative platform for analyzing multiple enzymes associated with central metabolic pathways of Saccharomyces cerevisiae using ultra fast mass spectrometry, J Biosci Bioeng. 2015;119: A Practical Introduction to Targeted Proteomics Fumio Matsuda* * fmatuda@ist.osaka-u.ac.jp Osaka University, Yamadaoka 1-5, Suita, Osaka , Japan (Received: April 20, 2016; Revised: May 26, 2016; Accepted: May 27, 2016) Targeted proteome analysis is an approach for quantitative analysis of multiple target peptides using liquid chromatography-triple quadrupole mass spectrometry. Although it is essentially similar to that of quantitative analysis of small molecules, several specific techniques are required for targeted proteome analysis including sample preparation methods, handling of nano-lc, and construction of SRM assay methods. Recent progresses in the sample preparation protocols, improved stability of modern nanolc machines, and development of softwares enable a wide application of the targeted proteome analysis in biological studies. Keywords: nano LC-MS; sample preparation; SRM assay;.targeted proteome analysis.