Development of a Human Cell-Free Expression System to Generate Stable-Isotope-Labeled Protein Standards for Quantitative Mass Spectrometry

Transcription

1 Development of a Human Cell-Free Expression System to Generate Stable-Isotope-Labeled Protein Standards for Quantitative Mass Spectrometry Ryan D. omgarden 1, Derek aerenwald 2, Eric Hommema 1, Scott Peterman 1, and John C. Rogers 1 1 Thermo Fisher Scientific, Rockford, IL; 2 Thermo Fisher Scientific, San Jose, C; 2 University of Iowa, Iowa City, I; Thermo Fisher Scientific, Cambridge, M

2 Overview Purpose: To develop a human cell-free expression system for the production of stableisotope-labeled (i.e. heavy) proteins. Methods: HeLa cell lysates, supplemented with 1 C 6 N 2 L-Lysine and 1 C 6 N 4 L- arginine,were used for in vitro translation (IVT) of recombinant fusion proteins. Labeled proteins were purified using affinity chromatography for mass spectrometry (MS) analysis. Results: We developed a novel, human-cell-based IVT system which expresses heavy proteins with 9-97% stable isotope in less than eight hours. Introduction Stable-isotope-labeled peptides are routinely used as internal standards for the quantification of enzymatically-digested protein samples. Sable-isotope-labeled proteins are ideal for MS sample preparation standardization. 1 Traditional in vivo expression systems, such as N-labeled E. coli or SILC, have been used to express recombinant heavy proteins. However, these systems are limited in their expression of toxic or insoluble proteins, require two to three days for protein expression, and may have low yield of functional (i.e. properly folded) proteins. In addition, because in vivo systems use stable-isotope-labeled cell lines, all proteins in the cell are isotopically labeled leading to significantly higher waste and cost. n alternative method to in vivo protein expression, in vitro translation (IVT), uses a cellular-extract system to transcribe DN into mrn, which is subsequently translated into protein. Most IVT systems utilize prokaryotic (e.g. bacteria) or non-human eukaryotic (e.g. rabbit reticulocyte) cell extracts. 2 However, these systems lack the components needed for proper folding and modification of human proteins, have lower expression yields, and inefficiently incorporate stable-isotope-labeled amino acids. In this study, we describe a novel human cell-free system,4 to express stable-isotopelabeled protein standards (Figure 1) as controls for sample-preparation loss, for digestion-efficiency determination, and as quantification standards. Methods Sample Preparation Protein expression and purification: For each gene, full-length cdns were expressed as C-terminal fusion proteins using the Thermo Scientific 1-Step Heavy Protein IVT Kit. Depending on the C-terminal affinity tag, expressed proteins were purified using a Thermo Scientific Pierce GST Spin Purification Kit and/or a Thermo Scientific HisPur Cobalt Purification Kit. Purified protein samples were separated by SDS-PGE and stained using Thermo Scientific Pierce GelCode lue Stain Reagent. Gel slices containing each protein were destained, reduced, and alkylated before digestion to peptides using trypsin for 4-16 hours. fter digestion, the peptides were desalted using Thermo Scientific C18 Stage tips and reconstituted with.1% TF. Expression of isotopically labeled proteins was performed in IVT reactions using a custom amino acid mix supplemented with the stable-isotope-labeled amino acids 1 C 6 N 2 L-Lysine and 1 C 6 N 4 L-arginine. ll reactions were incubated at C for 8 16 hours unless otherwise noted. Recombinant HIS-GFP protein fluorescence was measured using a GFP standard curve with a Tecan Safire fluorometer. LC-MS/MS nalysis NanoLC-2D high-pressure liquid chromatograph (HPLC) with a Thermo Scientific PepMap C18 column (7 µm ID x 2 cm) was used to separate peptides using a 4% gradient (: water,.1% formic acid; : acetonitrile,.1% formic acid) at a flow rate of nl/min for 4 min. Thermo Scientific LTQ Orbitrap XL ETD mass spectrometer was used to detect peptides using a top-six experiment consisting of single-stage MS followed by acquisition of six MS/MS spectra with collision-induced dissociation (CID) to aid in protein identification. Data nalysis MS spectra were searched for matches with a custom human SWISSProt database using Thermo Scientific Proteome Discoverer software version 1., and the SEQUEST search engine. Static modifications included carbamidomethyl with methionine oxidation. Lysine-8 and arginine-1 were used as dynamic modifications. SILC ratios were based on the area under the curve (UC) for each heavy and light peptide, and to determine stable-isotope. 2 Development of a Human Cell-Free Expression System to Generate Stable-Isotope-Labeled Protein Standards for Quantitative Mass Spectrometry

3 FIGURE 1. Heavy recombinant protein expression, purification and MS analysis. ) IVT lysates were combined with the reaction mixture, vector DN and stableisotope-labeled amino acids to express recombinant proteins. ) Expressed proteins were then purified and digested into peptides for LC-MS analysis. FIGURE. Expression of heavy mam of six different GST-fusion proteins e (* indicates an anti-gst cross-reactin isotope from peptides Purification Expressed Protein Purified Protein Harvest Cells Digestion LC-MS Cell Lysate IVT Expressed Protein Reaction Mixture: - stable-isotope-labeled amino acids - accessory proteins - energy mix, nucleotides, salts Isotope Incorporation Determination Figure 2. Heavy GFP protein expression. ) GFP was expressed for 2 hrs with increasing amounts of heavy arginine (rg1) and lysine (Lys8), and analyzed using the workflow described in Figure 1. ) GFP expression over time showing corresponding heavy-isotope. 1% 8% Heavy mino cid Titration FIGURE 4. MS spectra of peptides de spectra of stable-isotope-labeled G of stable-isotope-labeled cyclin D1 p Relative bundance 1 GFP D Cyclin D1 R p p [ ] Intensity [counts] GHLQGGk y4? 4.4 y7²? b? 4.28 y6? y 71 Percent Isotope Incorporation 1 8 6% 4% 2% % mino cid Concentration (mm) GFP Expression Time Course 1 8 rg1 Lys R= R=674 R=164 R=424 R= Intensity [counts] (1^) YPD Concentration (µg/ml) 6 6 % Heavy Relative bundance b2? y? R= Incubation time (hours) R=414 R=4624 R=484 R=4 R= Thermo Scientific Poster Note PN6614_E 6/12S

4 FIGURE. Expression of heavy mammalian proteins. ) nti-gst Western blot of six different GST-fusion proteins expressed using human IVT extract (* indicates an anti-gst cross-reacting band in the lysate). ) Table showing isotope from peptides derived from the proteins shown in. Sample MW % Heavy GFP 27 kda 9% D 19 kda 92% CyclinD1 6 kda 97% * p R kda 11 kda 91% 96% GFP D Cyclin D1 R p GPDH GPDH 7 kda 94% FIGURE 4. MS spectra of peptides derived from proteins shown in. ) MS spectra of stable-isotope-labeled GPDH peptide GHLQGGk. ) MS spectra of stable-isotope-labeled cyclin D1 peptide YPDNLLNDr. 1 p [ ] GHLQGGk 49.6 R= y7²? Intensity [counts] 2 1 y4? 4.4 b? y6? y7? b9? >9% isotope Relative bundance R= R= R=6424 R= R= R= R= R=674 R=224 R=164 R=424 R=64 R=494 R=484 R= m /z Heavy C YPDNLLNDr R=421 D 8 Relative bundance Intensity [counts] (1^) b8? 8.44 y9? 17.6 b2? y? y1²? y6? y7? y8? R=84 >9% isotope R= R= R= R=414 R=4624 R=484 R=44 R=4 R=4774 z= m /z Heavy 4 Development of a Human Cell-Free Expression System to Generate Stable-Isotope-Labeled Protein Standards for Quantitative Mass Spectrometry

5 GST Western blot VT extract Table showing s shown in. FIGURE. Heavy D protein-protein interaction. ) Coomassie-stained SDS- PGE gel of recombinant light and heavy D-GST-H-6xHIS purified from HeLa IVT lysates (L), using glutathione resin (E1) and cobalt resin (E2) tandem affinity. Flow troughs (FT) from each column are also indicated. ) Schematic of D phosphorylation and protein interactions during cell survival and cell death (i.e. apoptosis). C) D protein sequence coverage showing identified kt1 consensus phosphorylation sites (red box). D) MS spectra of stable-isotopelabeled D peptide HSSYPGTEDDEGmGEEPSPFr. e MW % Heavy 27 kda 9% 19 kda 92% D-GST-H-HIS Heavy D-GST-H-HIS 1 6 kda 97% kda 11 kda 7 kda 91% 96% 94% D 14--σ n in. ) MS k. ) MS spectra L FT1 E1 FT2 E2 L FT1 E1 FT2 E2 kt1 >9% isotope D P P 14-- D cl-2 + D ax = R=224 R=644 C Cell survival Cell death R= R=84 >9% isotope D Relative bundance HSSYPGTEDDEGmGEEPSPFr Intensity [counts] (1^) y? b17²? b16²? y6? b19²? y? b21²? b1? y4? b12? 2 y²? b16? b17? R= R= R=4774 >9% isotope R= R= R= R=44 z= R=4414 R= R=4674 R=4824 R=47 R=4264 R=414 R =74 R=44 R= m /z Heavy Thermo Scientific Poster Note PN6614_E 6/12S

6 Results Two different approaches for heavy-protein production were investigated. One used heavy SILC-labeled cells to produce a heavy-labeled IVT extract. The other method used normal light IVT extracts supplemented with heavy amino acids. lthough both methods successfully expressed GFP with stable-isotope of greater than 9%, the light lysates had a significantly higher level of protein expression and lower cost of production (Figure 2). Titration and time-course experiments using the light lysate with heavy amino acids showed that amino acid concentrations greater than or equal to 1 mm and incubation times longer than four hours were necessary for optimal protein expression and stable-isotope (Figure 2 and 2). In order to validate the use of this human heavy IVT system for production of humanbased proteins, six additional recombinant proteins were expressed and purified using GST or 6xHIS affinity purification. lthough all proteins were expressed as indicated by a Western blot (), only four of the six proteins were recovered after purification and sample preparation with high yield. s determined by our MS analysis of heavy and light peptides, all expressed proteins had stable-isotope equal to or greater than 9% (Figure and Figure 4). Ideal protein standards are identical to their endogenous counterparts. Expression of recombinant proteins in human cell-free extract systems has been shown to aid in proper protein folding and post-translational modification. 2 During the purification of one mammalian protein, D, we observed co-purification of light 14--σ with the heavy protein (Figure ). This protein-protein interaction is known to be mediated by 14-- binding of serine/threonine phosphorylation motifs (Figure ). MS analysis of the IVT-expressed protein identified three of four kt consensus phosphorylation sites (Figure 6C). Overall, these results indicate that recombinant D expressed using this human cell-free expression system has functional protein modifications and interactions. Conclusions Six different stable-isotope-labeled proteins were produced using a modified non- SILC human IVT system. Isotope efficiency of greater than 9% was observed for IVT reactions containing stable-isotope amino acids at concentrations greater than or equal to mm and incubated for longer than four hours. Co-purification of heavy D protein with light 14-- proteins suggests proper protein folding and post-translational modification of in vitro expressed protein. References 1. Hanke, S., et al. bsolute SILC for accurate quantitation of proteins in complex mixtures down to the attomole level. J. Proteom Res, 27, 7: Ciccimaro E., et al. bsolute quantification of phosphorylation on the kinase activation loop of cellular focal adhesion kinase by stable isotope dilution liquid chromatography/mass spectrometry. nal. Chem. 29, 81(9):4-1.. Mikami, S., et al. human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expr. Purif. 28, 62(2): Stergachis,., et al. Rapid empirical discovery of optimal peptides for targeted proteomics. Nat. Meth. 211, 8(12): Tzivion G, et al proteins; bringing new definitions to scaffolding. Oncogene. 21, 2(44):61-8. Safire is a trademark of Tecan. SEQUEST is a registered trademark of the University of Washington. ll other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 6 Development of a Human Cell-Free Expression System to Generate Stable-Isotope-Labeled Protein Standards for Quantitative Mass Spectrometry

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