Increasing Throughput in Targeted Proteomics Assays: 54-Plex

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Increasing Throughput in Targeted Proteomics Assays: 54-plex Quantitation in a Single Mass Spectrometry Run Robert A. Everley, Ryan C Kunz, Fiona E. McAllister, and Steven P. Gygi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac400845e • Publication Date (Web): 10 May 2013 Downloaded from http://pubs.acs.org on May 11, 2013

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Analytical Chemistry

Increasing Throughput in Targeted Proteomics Assays: 54-plex Quantitation in a Single Mass Spectrometry Run

Robert A. Everley*, Ryan C. Kunz, Fiona E. McAllister, Steven P. Gygi* Department of Cell Biology, Harvard Medical School, Boston, MA 02115, United States Abstract Targeted proteomics assays such as those measuring endpoints in activity assays are sensitive and specific but often lack in throughput. In an effort to significantly increase throughput, a comparison was made between the traditional approach which utilizes an internal standard and the multiplexing approach which relies on isobaric tagging. A kinase activity assay was used for proof of concept, and experiments included 3 biological replicates for every condition. Results from the two approaches were highly similar with the multiplexing showing greater throughput. Two novel 6-plex isobaric tags were added for a total of three 6-plex experiments (18-plex) in a single run. Next, three mass variants of the target peptide were labeled with the three isobaric tags giving nine 6-plex reactions for 54-plex quantitation in a single run. Since the multiplexing approach allows all samples to be combined prior to purification and acquisition, the 54-plex approach resulted in a significant reduction in purification resources (time, reagents etc.) and a ~50 fold improvement in acquisition throughput. We demonstrate the 54-plex assay in several ways including measuring inhibition of PKA activity in MCF7 cell lysates for a panel of 9 compounds.

* Corresponding authors: [email protected] & [email protected]

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Introduction Targeted quantitative proteomics assays using mass spectrometry offer a means of determining the abundance of key proteins under conditions of interest. The traditional approach, in which an isotopically labeled ‘heavy’ peptide of identical sequence as the target analyte peptide is used as a measure of the analyte’s abundance [1], has gained wide spread use in various applications including signal transduction [2, 3] and clinical specimens [4, 5]. Although targeted quantitative proteomics assays are sensitive and specific, they often lack in throughput, hindering their use in high throughput screening, clinical diagnostic and biomarker verification assays where sample requirements can be in the thousands. Moreover, as costs of purchasing and maintaining a mass spectrometer and demand for instrument time can be high, efficient use of instrument time is an important goal. Typically used in non-targeted assays, isobaric tagging approaches such iTRAQ [6] and TMT [7] enhance throughput by multiplexing six or more samples per tag. Comparisons amongst many samples can be achieved if one of the channels is dedicated to a common reference sample e.g. a control or pooled sample. The goal of this work was to significantly increase the throughput of a targeted proteomics assay by combining two newly synthesized tandem mass tag sets and three distinct mass variants of the target peptide. Kinase inhibition assays for Abl, AMPK, RSK and PKA, all kinases playing a role in certain cancers [8-11], were used as proof of concept. Methods Kinase Reactions For the in solution reactions using commercial kinase, 2.5 - 5 ng of kinase (SignalChem, Richmond, BC, CA), 10 µM ATP, 7.5 mM magnesium acetate, 5 µM substrate peptide and various concentrations of inhibitor (specified in each Figure) were added to a final volume of 50 µL in PBS pH=7.4 and allowed to react for 1hr at room temperature. For the traditional assay, the kinase reaction was quenched with the addition of 100 µL 1% TFA and a heavy internal standard peptide (Cell Signaling Technology, Danvers, MA) was added. For reactions in lysate, 5 µg of protein in a non-denaturing buffer (10 mM potassium phosphate buffer pH 7.0, 5 mM


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EGTA, 2 mM DTT, 10 mM MgCl2, 50 mM β-glycerolphosphate, 1 mM sodium vanadate, protease inhibitor, 0.5% NP-40, 0.1% Brij 35, 0.1% deoxycholic acid) was used as the kinase source and 50 µM ATP was added to a final volume of 50 µL containing 5 µM substrate. Protein concentration was measured using the Bradford assay. TMT Labeling For TMT labeling, the pH of the kinase reaction solution (50 µL) was adjusted to near 8.5 using 1M HEPES (5 µL) and acetonitrile (6 µL) was added to 10% v/v. Each reaction received 60 µg of TMT reagent for 250pmol of substrate and was allowed to react for 1hr at room temperature. The reaction was quenched for 15 min by the addition of 5 mM lysine in 50 mM ammonium bicarbonate. For the 54-plex assay, the substrate peptides were pre-labeled with TMT in batch (enough for 20 kinase reactions) at 10X concentration (50 µM) and aliquoted as necessary to minimize steps in the workflow. The pre-labeling strategy should be tested prior to use on other substrates. Purification of peptides for Mass Spectrometry (MS) analysis After the kinase reaction, the in-solution samples were purified using tC18 Sep-Pak Cartridges (Waters, Milford MA). For reactions in lysate, titanium dioxide enrichment was performed after the reaction as described [12]. Briefly, 2 mg of TiO2 beads (GL Sciences Inc., Torrance, CA) were added to peptides that had been purified via Sep-Pak cartridge. The sample was brought up in 90 µL of 2M lactic acid (Avantor Performance Materials, Center Valley, PA) in 50% acetonitrile. After shaking for one hour, the beads were washed with lactic acid, then with 50% acetonitrile containing 1% TFA, and eluted with 50mM dibasic potassium phosphate (pH =10) and purified on a Sep-Pak cartridge. After Sep-Pak purification, the eluent was dried in a speed-vac and 1/50th of the in solution samples and ½ of the TiO2 enriched samples were injected onto the mass spectrometer. Data acquisition and analysis A Famos autosampler (Dionex, Sunnyvale, CA) and Accela 600 pump (Thermo Scientific, San Jose, CA) connected to a 20 cm long, 100 µm id C18 column was used for analysis. The TMT labeled samples were acquired for 1hr on a Velos Pro Orbitrap (Thermo) 3 ACS Paragon Plus Environment


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using an MS3 method to eliminate interference [13]. The MS method was targeted, having a narrow m/z range (approximately ± 15 m/z from the lowest and highest m/z targeted) during the MS1 scan to minimize potential matrix affects due to co-eluting species and an inclusion list to focus the CID and HCD scans. No dynamic exclusion was used in order to increase the number of scans across each chromatographic peak (increasing the likelihood of quality MS3 spectra being obtained), and to prevent missing values. The traditional assay samples were acquired using an Exactive MS (Thermo) in full-scan mode, and the data were processed using Pinpoint software (Thermo). In all plots shown below, the error bars represent the standard error of the mean. Results and Discussion Traditional vs. Multiplexed assays To examine the potential merit of using isobaric tagging to increase throughput in targeted proteomics assays, a direct comparison was made with the traditionally used approach, which calculates the ratio of an analyte peptide’s abundance to that of an isotopically labeled internal standard peptide of the same sequence. As a proof of concept, an in vitro kinase assay monitoring the formation of phosphorylated substrate upon incubation with ATP and kinase was used for this study. Six kinase reactions (three control vs. three inhibitor treated) were performed for both the multiplexed and the traditional assays (Figure 1A). After one hour incubation, the multiplexing samples were labeled with isobaric tagging reagents and the traditional assay samples were quenched and a heavy internal standard peptide was added. Before purification using a reversed phase SPE cartridge, the six multiplexed reactions were combined into a single sample, while the traditional workflow required that each reaction be individually purified for a total of six purifications and six injections on the mass spectrometer (MS). In contrast, the multiplexed workflow required only a single injection on the instrument and since all six tags are isobaric, only one target precursor m/z was monitored in the MS. The resulting data were nearly identical, with both assays revealing a greater than 60% reduction of Abl kinase activity in the presence of 250 nM staurosporine (Figure 1B). Data from the traditional approach can easily be compared to other experiments since the analyte data is held relative to an internal standard which is commonly added to all samples. However, 4 ACS Paragon Plus Environment

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comparisons amongst experiments can also be achieved with the multiplexing approach provided a common control sample is included and the subsequent data are held relative to this control. The control sample could be a wild type or untreated sample, or a pooled sample from a cohort of healthy patients etc. For the purposes of the kinase assays used in this study, the 126 channel was always a non-treated control representing 100% kinase activity. The remaining channels were either control replicates or inhibitor treated. The experiment in Figure 1B was duplicated for AMPK kinase (Figure 1C). Staurosporine inhibits AMPK more potently than Abl, eliminating nearly all kinase activity at 250 nM. As with the Abl kinase assay, the data between the AMPK multiplexed and traditional internal standard based assays were in good agreement. 18-plex Quantification Beyond six-plex quantitation in a single run using a single TMT tag, two novel TMT tags were added to increase the throughput 3X to 18-plex. As in Figure 1, where a 3 vs. 3 experiment was conducted for a single inhibitor, now three inhibitors can be monitored in triplicate in a single run (Figure 2A). The three TMT tags have distinct masses as a result of their structural differences as shown in Figure 2B. The light tag is the commercially available TMT 6-plex tag, while the novel medium and heavy versions incorporate an aminopropanoic and aminobutyric acid insertion (shown in red), respectively. The resulting medium tag is 71.02 Da (C3H5NO) heavier than the light tag and the heavy tag is 14.02 Da (CH2) heavier than the medium tag. The three TMT tagged cognates have similar ionization efficiency but unlike triple labeled SILAC cognates, they typically do not co-elute. Three distinct masses per peptide result in three targets during MS analysis with each reporting six distinct experiments for eighteen experiments in total (Figure 2C). Although used at a high concentration (250 nM) to reveal off-target effects, inhibition of RSK kinase with the EFGR inhibitor AG1478 is still less than 40%, demonstrating the specificity of the inhibitor (Figure 2D). In contrast, the PKC inhibitor Go6976 inhibits RSK nearly as well as staurosporine, a non-specific yet strong inhibitor of numerous kinases. Previous studies have shown that although designed for PKC inhibition, many bisindoylmaleimide compounds potently inhibit RSK [14]. Experiments using Go6976 should be interpreted with caution as PKC inhibition alone may not be responsible for the observed phenotypes [15]. 5 ACS Paragon Plus Environment


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In addition to achieving 18-plex quantitation by using three distinct 6-plex isobaric tags [16], another option is using a single 6-plex tag with three distinct mass variants of the target peptide. One way to achieve this would be to combine triple labeling SILAC [17] with a single isobaric tag as demonstrated by Dephoure et al. [18]. To further extend throughput capabilities, a combination of the three distinct TMT tags and three mass variants of the target peptide was implemented. This created a 3 x 3 matrix of three versions of the analyte peptide × three versions of the isobaric tag, resulting in nine, 6-plex reactions for a 54-plex experiment (Figure S1). To demonstrate this, five examples of 54-plex analyses in single runs are discussed below. 54-plex Quantification Three variations (Light, Medium and Heavy) of the substrate peptide combined with the three TMT tags discussed above were integrated for a 54-plex analysis, where an 18-point IC50 curve was performed in triplicate in a single run. This experiment was performed both in solution using purified PKA (Figure 3A) and in lysate from the ER-positive breast cancer cell line MCF7 (Figure 3B), in which PKA plays a role in tamoxifen resistance [11]. The cells were lysed in non-denaturing lysis buffer to preserve enzyme activity and conformation. Three mass cognates of the PKA substrate were utilized with the light peptide differing from the medium by a minor sequence variation (PFR for FK) at the far C-terminal end of the peptide and the heavy peptide differing from the medium peptide by an isotopically labeled phenylalanine (Figure 3C). Having three peptide mass variants and three TMT tags, nine targets were monitored after only a single purification step (Figure 3D). While the three variations of the substrate peptide could be isotopically labeled versions of the same peptide sequence e.g. triple labeling SILAC, in the case of kinase substrates, as long as the kinase recognition motif remained constant, minor sequence variants were possible. For PKA, the recognition motif is RRx[S/T]#, where x is any amino acid and # is preferentially a hydrophobic residue. All three versions contained in common two amino acids N-terminally and three C-terminally adjacent to the kinase recognition motif: NKRRGsVPIL where the serine is phosphorylated. Each peptide sequence was used for one of three replicates of the 18-point curve. The different peptides show similar response as evident by the size of the error bars on each data point. To further examine the similarity of the light, medium and heavy peptides as substrates, the in-solution data was used to calculate each IC50


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individually. The values were: 15.1 nM (light peptide), 14.2 nM (medium peptide) and 18.7 nM (heavy peptide) giving a mean IC50 of 16.0 nM with a reasonable standard error of 1.4 nM. While performing inhibition assays in solution using purified kinases is common, using lysates can be advantageous and was demonstrated recently in the assay known as KAYAK [2, 19-20]. Lysates contain biologically relevant small molecules (e.g. lipids and metabolites) which may play unknown roles in the kinase pathway of interest. Also inhibition takes place in the presence of other kinases and proteins that could interact with the inhibitor. Finally, if designing an inhibitor to target a specific illness such as breast cancer, using breast cancer cell or tissue lysate during screening is more relevant than using a purified recombinant kinase in solution as competitors may vary in abundance in a tissue specific manner. The Gold Standard assay for inhibitor screening is a filtration binding assay with either fluorescent or radioactive readout [21]. The filtration binding assay would likely not be successful in lysate due to its lack of specificity as other peptides and proteins may be phosphorylated during the reaction. In this study it was observed that lysate IC50 values were typically 5-10X higher than in solution. Conducting the reaction in lysate does provide one additional challenge, as the lysate is in a detergent that should ideally be removed prior to MS analysis. This is done as a byproduct of the titanium dioxide (TiO2) enrichment step. This step allows complete removal of the detergent and has the added benefit of depleting the non-phosphorylated substrate peptide which is often high in abundance and signal intensity. With the traditional approach, fifty-four individual TiO2 enrichments with fifty-four reversed phase SPE purifications before and after the enrichment would be required. But with the 54-plex assay described here, only a single TiO2 enrichment is needed combined with a single reversed phase SPE step (before and after enrichment) to prepare the sample for MS analysis. When pursuing compounds of interest as potential therapeutics, not only is the potency (IC50) towards its target important, but the mechanism of action (MOA) detailing how the inhibitor functions is also key. By performing three six-point curves in triplicate at three different ATP concentrations, the IC50 and MOA of an inhibitor can be determined in a single run. Conducting each measurement in triplicate provides statistical confidence and the ability to remove outliers without repeating the experiment. Two inhibitors with similar potency yet distinct MOAs were compared as proof of principle. Triplicate IC50 curves ranging from 0-5 µM 7 ACS Paragon Plus Environment


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inhibitor were performed in the presence of 0.01, 1 and 10 mM ATP and commercial PKA (Figure S2). PKI’s IC50 from the six point curve at 0.01 mM ATP agrees well with the 18-point curve above (Figure 3A) which used the same ATP concentration. PKI’s efficacy is insensitive to ATP concentration, indicative of a non-ATP competitive inhibitor (Figure S2-A). In contrast, the IC50 of staurosporine increases 20X from low to high ATP concentration, indicative of an ATP-competitive inhibitor (Figure S2-B). While in Figure 3, each data point was the average of data obtained from the light, medium and heavy peptides, in Figure S2, each data point is the average of data from the light, medium and heavy isobaric tag. In addition to sample-sample variability, the height of the error bars also represents variability associated with using the three different labels. As seen from the height of the error bars, the data from the three different tags typically yield similar results. Analogous to the 6 and 18-plex experiments in Figures 1 and 2 respectively, Figure 4 depicts a nine inhibitor, 54-plex experiment from a single mass spectrometry run. MCF7 lysate was the source of PKA activity, and the nine inhibitors tested were at a relatively high concentration (5 µM) to detect off-target effects. Many of the inhibitors (e.g. the EGFR inhibitors) showed no off-target inhibition of PKA activity. A recent study of off-target effects for 178 inhibitors, found both AG1478 and Getfinib in the top 10% of the most unispecific inhibitors in their panel [22]. Staurosporine (6) and PKI (8) showed the greatest inhibition as expected. The strong inhibition by PKI lends credibility to the specificity of the kinase assay even in the presence of a complex lysate, as PKI is a selective, substrate-competitive inhibitor. PKI mimics PKA substrates by having the PKA recognition motif with the exception of a phosphorylatable residue i.e., RRNAI instead of an RRXS/T# where # = a hydrophobic residue. The two adjacent arginines on PKI form ion pairs with conserved glutamic acid residues on PKA’s catalytic subunit while the isoleucine of PKI interacts with the kinase via a combination of hydrogen bonding and hydrophobic interaction [23]. In terms of off-target effects, the PKC inhibitor Ro318425 showed the most off-target inhibition. However this inhibition (with 40% of activity remaining) was relatively modest compared to Go6796 off-target inhibition of RSK, where 9% of kinase activity remained (Figure 2). Because in a lysate inhibition could be indirect, i.e., by inhibiting an upstream kinase that activates PKA rather than PKA directly, a Ro318325 inhibition experiment was performed in solution using purified PKA (Figure S3).


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Direct inhibition in solution with 0.5 µM inhibitor was observed but still at a relatively modest amount (40% activity remained) for this inhibitor concentration. Other combinations of the 54-plex are possible such as combining nine, 2 vs. 2 vs. 2 sixplex reactions which could be used to screen two drugs and a control in duplicate for a total of eighteen drugs per run. In cases where n=1 is acceptable, nine sets of a single control and five compounds could be performed for 45 drugs in a single run. Replicates could then be achieved by additional injections onto the mass spectrometer. The creation of multi-mass variants of the peptide of interest in higher order multiplexing, also known as hyperplexing [18] experiments, can be achieved by combining isobaric tagging with not only metabolic labeling strategies such as SILAC [17] and 15N labeling [24], but also with other chemical labeling strategies such as labeling cysteines [25], carboxylic acid groups [26] or by digestion in 18O water [27] etc. The chemical labeling strategies are particularly attractive in the case of tissue samples or body fluids where metabolic labeling can be challenging and costly. Although the data demonstrated herein was relative quantitation, absolute quantitation using isobaric tagging reagents is possible by dedicating one [28] or more [29] channels to synthetic peptides of known concentration. By extending the method described here to ten-plex isobaric tags [30], the throughput could be further enhanced to 90-plex without increasing the complexity of the MS1 scan. This would allow the analysis of nearly an entire 96-well plate in a single LC/MS run. The sample preparation protocol presented here is relatively straightforward and should be amenable to robotic implementation. Conclusions The traditional internal standard assay for targeted proteomics was multiplexed using isobaric tagging with no loss in data quality and without the need of an internal standard. By creating multi-mass variants of the target peptide and combining them with three isobaric tags of differing mass, a 54-plex targeted proteomic analysis was achieved in a single run resulting in an improvement in throughput of approximately 1.5 orders of magnitude. In addition to in vitro kinase assays, the throughput enhancement shown here has potential application to other AQUA based targeted methods. The multiplexing of 54 samples required that only nine target masses be monitored, which is by no means the upper limit achievable by modern mass spectrometry instrumentation. Besides minimizing time, reagents and consumables during the purification 9 ACS Paragon Plus Environment


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step by collapsing many samples into one, these improvements in throughput allow a more efficient use of the mass spectrometer saving costly instrument time and making high sample volume projects (n >1000) more feasible while still maintaining the sensitivity and specificity that mass spectrometry provides. Acknowledgements The authors would like to thank John Rogers of Thermo Fisher Scientific and Karsten Kuhn and Ian Pike of Proteome Sciences for synthesizing the medium and heavy isobaric tagging reagents. We would also like to thank Jeffrey Knott and John Rush from Cell Signaling Technology for help with synthesis of the kinase substrate peptides. The insightful comments from the reviewers were greatly appreciated. This research was partially funded by a grant from the NIH (GM67942 and HG3456) to SPG.


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A

C

B

Figure 1. Multiplexing a targeted in vitro, peptide-based kinases for six reactions (3 control vs. 3 inhibitor). Two commercial kinases inhibited by staurosporine (250 nM), were used to compare the Multiplexing and Traditional assays (A). Although the Multiplexing protocol required 6x less purification columns and MS injections, the data across three replicates were highly similar for both Abl (B) and AMPK (C). The core substrate peptide sequence recognized by the kinase is underlined in the legend with the phosphorylated residue in bold. Error bars represent ± S.E.M. for n=3.



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A

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B

D

C

Figure 2. An 18-plex in vitro kinase assay. Using commercial RSK kinase, a triplicate analysis of control vs. a single inhibitor concentration was performed, but now comparing three different inhibitors in a single 18plex run (A). Throughput is further enhanced by the addition of two novel TMT reagents giving in total three distinct TMT tags, each with a unique m/z (B). The three targets are monitored in a single run (C) depicting the various specificities of the inhibitors (D). The core substrate peptide sequence recognized by the kinase is underlined in the legend with the phosphorylated residue in bold. Go6796 showed significant off-target inhibition.


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A

B

PKI Inhibition of PKA in Solution (n=3)

1.2

PKI Inhibition of PKA in MCF7 Lysate (n=3)

1.4

IC50 = 16 nM

IC50 = 75 nM

1.2

Relative Product Formation

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Relative Product Formation

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0.8

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PKI Concentration ( M)

PKI Concentration ( M)

Light Medium Heavy

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Inhibitor Concentration (µ µM) 0 0.00025 0.0025 0.025 0 0.00025 0.0025 0.025 0 0.00025 0.0025 0.025 126 127 128 129 Light TMT

Light Peptide NKRRGSVPILPFR

Medium Peptide NKRRGSVPILFK

NKRRGSVPILx + PKA + ATP

0.25 0.25 0.25 130

2.5 2.5 2.5 131

0 0 0 126

0.0005 0.0005 0.0005 127

0.005 0.05 0.005 0.05 0.005 0.05 128 129 Medium TMT

D

Heavy Peptide NKRRGSVPILF#K

NKRRGpSVPILx

54-plex

0.5 0.5 0.5 130

5 5 5 131

0 0 0 126

0.001 0.001 0.001 127

0.01 0.1 0.01 0.1 0.01 0.1 128 129 Heavy TMT

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10 10 10 131

Samples

Purifications

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54

1

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9

Figure 3. Two 54-plex analyses consisting of an 18-point IC50 curve performed in triplicate. Inhibition of PKA using the peptide inhibitor PKI was conducted in solution (A) using 2 ng of commercially available PKA, and in 5 µg of lysate from the breast cancer cell line MCF7 (B). In addition to 3 distinct TMT tags, three variants of the substrate peptide are now included (C) resulting in 9 target peptides (D). Residues 243-252 from a known in vivo PKA substrate, EPB42, were used to measure product formation. Lysate IC50 values were typically higher than those using purified kinase.



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Figure 4. A 54-plex screen of nine compounds in triplicate in a single run. For each of the 9 peptide forms, 3 control vs. 3 inhibitor treated samples were compared in MCF7 lysate (5 µg per reaction) at a single (5 µM) inhibitor concentration to examine potential off-target effects on PKA activity (see table). The core substrate peptide sequence recognized by the kinase is underlined in the legend with the phosphorylated residue in bold.


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References 1. Kirkpatrick, D.S.; Gerber, S.A.; Gygi, S.P. Methods 2005, 35, (3), 265-273. 2. Yu, Y.; Anjum, R.; Kubota, K.; Rush, J.; Villen, J.; Gygi, S.P. PNAS 2009, 106, (28), 11606-11611. 3. Prabakaran, S.; Everley, R.A.; Landrieu, I.; Wieruszeski, J.M.; Lippens, G.; Steen, H.; Gunawardena, J. Mol. Syst. Biol. 2011, 7, (482), 1-15. 4. Chen, Y.; Gruidl, M.; Remily-Wood, E.; Liu, R.Z.; Eschrich, S.; Lloyd, M.; Nasir, A.; Bui, M.M.; Huang, E.; Shibata, D.; Yeatman, T.; Koomen, J.M. J. Proteome Res., 2010, 9, (8), 4215-4227. 5. Kuzyk, M.A.; Smith, D.; Yang, J.C.; Cross, T.J.; Jackson, A.M.; Hardie, D.B.; Anderson, N.L.; Borchers, C.H. Mol. Cell. Proteomics, 2009, 8, (8), 1860-1877. 6. Ross, P.L.; Huang, Y.N.; Marchese, J.N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D.J. Mol. Cell. Proteomics, 2004, 3, 1154-1169. 7. Thompson, A.; Schafer, J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Neumann, T.; Hamon, C. Anal. Chem., 2003, 75, 1895-1904. 8. Srinivasan, D.; Plattner, R. Cancer Res., 2006, 66, 5648-5655. 9. Zhou, J.; Yang, Z.; Tsuji, T.; Gong, J.; Xie, J.; Chen, C.; Li, W.; Amar, S.; Luo, Z. Oncogene, 2011, 30, 1892-1900. 10. Nguyen, T.L. Anti-Cancer Agents in Med. Chem., 2008, 8, 710-716. 11. de Leeuw, R.; Flach, K.; Bentin Toaldo, C.; Alexi, X.; Canisius, S.; Neefjes, J.; Michalides, R.; Zwart, W. Oncogene, 2012, doi:10.1038/onc.2012.361. 12. Kettenbach, A.N.; Gerber, S.A. Anal. Chem. 2011, 83, 7635-7644. 13. Ting, L.; Rad, R.; Gygi, S.P.; Haas, W. Nat. Methods, 2011, 8, 937-940. 14. Davies, S.P.; Reddy, H.; Caivano, M.; Cohen, P. Biochem. J., 2000, 351, 95-105. 15. Koivunen, J.; Aaltonen, V.; Koskela, S.; Lehenkari, P.; Laato, M.; Peltonen, J. Cancer Res., 2004, 64, 5693-5701. 16. Huttlin, E.L.; Jedrychowski, M.; Kuhn, K.; Dai, C.; Ting, L.; McAlister, G.; Rad, R.; Rogers, J.C.; Pike, I.; Haas, W.; Gygi, S., Enhanced Isobaric Labeling Enables 18-plexed Quantitative Exploration of the HSF1-dependent Cellular Response to Multiple Proteotoxic Stresses at a Proteomic Scale. American Society for Mass Spectrometry Conference, Vancouver, CA, 2012. 17. Blagoev, B.; Ong, S.E.; Kratchmarova, I.; Mann, M. Nat. Biotechnol., 2004, 22 (9), 1139-1145. 18. Dephoure, N.; Gygi, S.P. Sci. Signal., 2012, 5 (217), rs2. 19. Kubota, K.; Anjum, R.; Yu, Y.; Kunz, R.C.; Andersen, J.N.; Kraus, M.; Keilhack, H.; Nagashim, K.; Krauss, S.; Paweletz, C.; Hendrickson, R.C.; Feldman, A.S.; Wu, C.L.; Rush, J.; Villen, J.; Gygi, S.P. Nat. Biotechnol., 2009, 27 (10), 933-940. 20. Kunz, R.C.; McAllister, F.E.; Rush, J.; Gygi, S.P. Anal. Chem., 2012, 84, 6233-6239. 21. Ma, H.; Deacon, S.; Horiuchi, K. Expert Opin. Drug Discov., 2008, 3 (6), 607-621. 22. Anastassiadis, T.; Deacon, S.W.; Devarajan, K.; Ma, H.; Peterson, J.R. Nat. Biotechnol. 2011, 29 (11), 1039-1045. 23. Bossemeyer, D.; Engh, R.A.; Kinzel, V.; Phonstingl, H.; Huber, R. EMBO Journ., 1993, 12 (3), 849859. 24. Oda, Y.; Huang, K.; Cross, F.R.; Cowburn, D.; Chait, B.T. PNAS, 1999, 96, 6591-6596. 25. Gygi, S.P.; Rist, B.; Gerber, S.A.; Turecek, F.; Gelb, M.H.; Aebersold, R. Nat. Biotechnol., 1999, 10, 994-999. 15 ACS Paragon Plus Environment


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54 Samples, 1 Injection Light Peptide

Medium Peptide

Heavy Peptide

Light TMT6 Medium TMT6 Heavy TMT6

Intensity

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Time

For TOC only.


Increasing Throughput in Targeted Proteomics Assays: 54-Plex

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