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Evaluation and Application of Dimethylated Amino Acids as Isobaric Tags for Quantitative Proteomics of TGF-#/Smad3 Signaling Pathway Qing Yu, Xudong Shi, Tyler Greer, Christopher B. Lietz, K. Craig Kent, and Lingjun Li J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00641 • Publication Date (Web): 26 Jul 2016 Downloaded from http://pubs.acs.org on July 26, 2016

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Journal of Proteome Research

Evaluation and Application of Dimethylated Amino Acids as Isobaric Tags for Quantitative Proteomics of TGF-β/Smad3 Signaling Pathway

Qing Yu,1 Xudong Shi,2 Tyler Greer,3 Christopher B. Lietz,3 K. Craig Kent,2 Lingjun Li1,3,*

1

School of Pharmacy, University of Wisconsin, Madison, WI 53705, USA

2

Department of Surgery, School of Medicine and Public Health, University of Wisconsin,

Madison, WI 53705, USA 3

Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA

*Address reprint requests to: Dr. Lingjun Li, School of Pharmacy & Department of Chemistry, University of Wisconsin, 777 Highland Ave, Madison, WI 53705. E-mail: [email protected]. Phone: (608)265-8491, Fax: (608)262-5345.

1 ACS Paragon Plus Environment


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Abstract Isobaric labeling has become a widespread tool for quantitative proteomic studies. Here, we report the development and evaluation of several dimethylated amino acids as novel isobaric tags for quantitative proteomics. Four-plex dimethylated alanine (DiAla), valine (DiVal) and leucine (DiLeu) have been synthesized, sharing common features of peptide-tagging and reporter ion production. DiAla and DiLeu are shown to achieve complete labeling. These two tags’ impacts on peptide fragmentation and quantitation are further evaluated using HEK cell lysate. DiAla labeling generates more abundant backbone fragmentation whereas DiLeu labeling produces more intense reporter ions. Nonetheless, both tags enable accurate quantitative analysis of HEK cell proteomes. DiAla and DiLeu tags are then applied to study TGF-β/Smad3 pathway with four differentially treated mouse vascular smooth muscle (MOVAS) cells. Our MS data reveal proteome-wide changes of AdSmad3 as compared to the GFP control, consistent with previous finding of causing smooth muscle cell (SMC) dedifferentiation 1. Additionally, the other two novel mutations on the hub protein Smad3, Y226A and D408H, show compromised TGF-β/Smad3-dependent gene transcription and reversed phenotypic switch. These results are further corroborated with Western blotting and demonstrate that the novel DiAla and DiLeu isobaric tagging reagents provide useful tools for multiplex quantitative proteomics.

Keywords: Dimethylated amino acid, isobaric tag, smooth muscle cell, quantitative proteomics, phenotype


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Introduction Quantitative proteomics using mass spectrometry (MS) has gained its popularity in its application to the relative quantification of protein expression levels in different biological states. Quantitative MS approaches are either label-free

2, 3

or label-based

4-6

. Labeling methods allow

multiplexing of samples and enable comparative analysis in one LC-MS run. Thus, labeling can increase throughput, improve quantitative accuracy, and decrease run-to-run variability. Numerous MS-based chemical derivatization approaches have been reported and widely used for quantitative proteomics

6-8

. Among them, isobaric labeling techniques have undergone rapid

development in recent years due to their capability for increased multiplexing 6. In addition, the multiplexing capacity of these reagents allows for the inclusion of more technical and biological replicates, providing greater statistical validation within any given experiment 9. Tandem mass tags (TMT)

10

and isobaric tags for relative and absolute quantitation

(iTRAQ) 11 are two major types of isobaric reagents that are commercially available. Our lab has developed a novel, cost-effective 4-plex set of dimethylated leucine (DiLeu) isobaric reagents and found its performance to be comparable to commercial reagents

12, 13

. Subsequent

development provided an array of DiLeu-based reagents in an attempt to meet different research requirements for relative

14

and absolute quantitation

15

. In this work, we explore the

development and application of other dimethylated amino acids for quantitative proteomics and compare their performance with the more established 4-plex DiLeu tags, in the context of restenosis. Restenosis is a common adverse event of endovascular procedures that are used to treat the vascular damage from atherosclerosis. It is characterized by the recurrence of stenosis, a narrowing of a blood vessel, leading to restricted blood flow. It occurs in 30-50% of patients



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depending upon the artery treated, imposing a major limitation to the long term success of surgical interventions 16, 17. Transforming growth factor beta (TGF-β) plays a prominent role in regulating a variety of cellular functions including cell migration, cell proliferation, apoptosis, differentiation, immunosuppression, inflammation, tumor suppression, and angiogenesis signaling also has been associated with several diseases

18

. Abnormal TGF-β

19, 20

, including restenosis

21

. The

prevailing view is that through its primary signal transduction mediator, Smad3, TGF-β stimulates vascular SMC intimal hyperplasia (IH) which is the main cause for restenosis. Activation of Smad3 by TGF-β stimulates IH and enhances adaptive remodeling. However, Smad3 serves as a hub protein for over 50 protein-protein interactions

22

so that simply

knocking out Smad3 is not a feasible treatment. Differentiating the TGF-β/Smad3 pathways that lead to adaptive remodeling versus IH will further clarify the underlying mechanisms behind restenosis as well as inform the design of inhibitors that have the ability to alter IH but not interfere with adaptive remodeling. Therefore, dissecting intertwined and probably overlapping binding sites on Smad3 is necessary to determine the specific pathways responsible for restenosis. Y226A and D408H are two mutations that can block or reverse Smad3 functions

23,

24

. These critical mutations provide ideal targets to achieve our desired goal. In this work, we expand upon the original DiLeu isobaric tagging concept by synthesizing

and testing two novel dimethylated amino acids (i.e., DiAla and DiVal) to provide more options in the amino-acid based isobaric reagent toolbox. Based on our evaluation, DiAla and DiLeu tags offer complete labeling. We then evaluate their impacts on labeled peptide fragmentation and discover that DiAla tags produce more backbone fragments compared to DiLeu. However, they both offer comparable quantitative accuracy. Furthermore, the average cost of a set of 4plex DiAla or DiLeu labels sufficient for a single experiment (100 µg of protein digest per channel) is estimated to be about ten dollars, whereas the same amount of iTRAQ or TMT 4 ACS Paragon Plus Environment

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reagents would cost more than several hundred dollars, due to complex, multi-step syntheses required to produce these commercial reagents. The reduced cost for reagent production with reliable quantitation accuracy makes amino-acid based reagents an attractive alternative for quantitative proteomics. To demonstrate their utility in large-scale investigation of systems biology problem, we employ these custom-synthesized tags in the proteome-wide analysis of changes caused by manipulation of Smad3 to help identify and establish TGF-β/Smad3 induced signal transduction pathway and differentiate IH from remaining overlapping pathways.

Experimental Procedures 1. Materials Reagents for the synthesis of labels included the following (Scheme S1): all isotopic amino acids (L-leucine and L-leucine-1-13C, valine-1-13C,

15

15

N, L-alanine and L-alanine-1-13C,

15

N, L-valine and L-

18

O water 97% (H218O) were purchased from Cambridge Isotope

N) and

Laboratories Inc (Tewksbury, MA). Heavy formaldehyde (CD2O), heavy water (D2O), sodium cyanoborodeuteride (NaBD3CN), sodium cyanoborohydride (NaBH3CN), 4-(4,6-Dimethoxy1,3,5-triazin-2-yl)-4-methylmorpholinium

tetrafluoroborate

(DMTMM)

and

1.0

M

triethylammonium bicarbonate buffer (TEAB) were purchased from Sigma-Aldrich. NMethylmorpholine (NMM) was purchased from TCI America (Tokyo, Japan). Protease (Cat. # 11836170001) and phosphatase inhibitor cocktail tablets (Cat. # 04906845001) were purchased from Roche. Tris base, urea, water, acetonitrile, and formic acid (FA) for UPLC were purchased from Fisher Scientific. 2. Synthesis of N, N-Dimethylated Amino Acids Synthesis followed procedures published previously Heavy isotopes, 2H,

13

C,

15

N,

12

with minor modifications (Scheme S1).

18

O are strategically placed onto the structure to create three

isobaric 4-plex reagent sets (Figure 1).



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3. Cell Culture HEK 293 and MOVAS cells were obtained from American Type Culture Collection (ATCC) and maintained in DMEM media containing 10% FBS at 37 oC with 5% CO2 as instructed. Biological triplicate MOVAS cells were then infected with retrovirus expressing wild-type or Smad3 point mutation (Y226A or D408H) and sorted by fluorescence activated cell sorting (FACS). The expression of wild-type or Smad3 mutants were confirmed by FACS and Western blotting. Biological triplicate MOVAS cells expressing GFP were used as control. 4. Enzymatic Protein Digestion Proteins were dissolved in 8 M urea, reduced (5 mM DTT, 1 h at room temperature) and alkylated (15 mM IAA, 30 min at room temperature in the dark). Alkylation was capped by incubation in 5 mM DTT for 5 min at room temperature. Urea was adjusted to 0.9 M and trypsin was added in a 1:50 (w/w) ratio and incubated for 18 h at 37°C. Each digest was quenched by the addition of TFA to a final concentration of 0.3%, desalted on a C18 SepPak cartridge (Waters, Milford, MA), and dried under vacuum. Protein digests were then re-dissolved in 0.5 M TEAB before labeling. 5. Activation of DiAla, DiVal, DiLeu and Protein Digest Labeling DiLeu tags were suspended in anhydrous DMF and combined with DMTMM and NMM at 0.6× molar ratios to DiLeu. The mixture was then vortexed at room temperature for 60 min. Following centrifugation, the supernatant was immediately mixed with protein digest. Peptides were labeled by adding the activated label solution at a 10:1 label to peptide digest ratio by weight and vortexing at room temperature for 2 h. The reaction was quenched by adding 5% NH2OH to the final concentration of 0.25% and dried under SpeedVac. Peptides labeled with different channels were then mixed with equal amounts or experiment-specific ratios (i.e. 8:4:2:1 and 1:2:4:8).


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6. Strong Cation Exchange (SCX) Fractionation Peptides labeled with different isotopic variants of isobaric tags were mixed together and resuspended in 100 µL of 75% 10 mM ammonium formate, 25% acetonitrile, pH 3.0 with formic acid and fractionated by strong cation exchange (SCX) liquid chromatography using a PolySULFOETHYL A column (200 mm × 2.1 mm, 5 µm, 300 Å, PolyLC, Columbia, MD). Mobile phase A consisted of 75% 10 mM ammonium formate, 25% acetonitrile, pH 3.0 with formic acid, and mobile phase B was 75% 500 mM ammonium formate, 25% acetonitrile, pH 6.8. The column was initially loaded and washed for 20 min with 0% B. Peptides were eluted using a linear gradient of 0–50% B over 50 min and then increased to 100% over 10 min. The column was subsequently washed at 100% B for an additional 10 min all at a flow rate of 0.2 ml/min. The column effluent was monitored at 280 nm with a Waters 2489 UV/Visible detector, and fractions were collected with a FC-4 fraction collector (Rainin Dynamax) followed by drying under SpeedVac. 7. Reversed-phase Separation and Mass Spectrometry 7.1. Normalized Collision Energy (NCE) Optimization NCE ramp tests were conducted by operating the Orbitrap Elite at a MS resolution of 30K, MS2 resolution of 15K, and an isolation width of 2 Th. The most abundant ion from m/z 350-1500 in each cycle was isolated 10 times and fragmented with HCD normalized collision energies (NCE) of 10, 15, 20, 25, 27, 30, 32, 35, 40, and 45. The Coon OMSSA (Open Mass Spectrometry Search Algorithm) Proteomic Analysis Software Suite (COMPASS)

25

was used for data

analysis by HCD energy comparisons. 7.2. Fragmentation Comparison between DiAla and DiLeu 4x50 µg of peptides labeled with 4-plex DiAla and 4-plex DiLeu were mixed together and fractionated. Peptide identification by liquid chromatography/tandem mass spectrometry



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(LC/MS/MS) analysis was performed using a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) with a NanoAquity UPLC system (Waters). Peptides were dissolved in 40 µL of 0.1% FA, out of which 3 µL was loaded and separated on a 75 µm x 15 cm homemade column packed with 1.7 µm, 150 Å, BEH C18 material obtained from a Waters UPLC column (part no. 186004661). 7.3. MOVAS Protein Identification and Quantification For each biological replicate, 4x50 µg of peptide digest from 4 samples were labeled with 4-plex DiAla or 4-plex DiLeu and fractionated. Peptide identification by LC/MS/MS analysis was performed using an Orbitrap Elite Ion Trap-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) interfaced with a NanoAquity UPLC system (Waters). Peptides from each fraction were dissolved in 40 µL of 0.1% FA, out of which 3 µL was loaded and separated on a 75 µm x 15 cm self-fabricated column packed with 1.7 µm, 150 Å, BEH C18 material obtained from a Waters UPLC column (part no. 186004661). 8. Western Blot Cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate) with protease inhibitor cocktail (Roche). Forty micrograms of protein from each sample was separated on SDS-PAGE, and then transferred to PVDF membranes. Proteins on transferred membrane were assessed by antibodies to smooth muscle alpha actin (SMA), calponin and myosin heavy chain. After washing with TTBS and incubated with horseradish peroxidase-conjugated secondary antibodies, the PVDF membrane was developed with chemiluminescence substrate (Fisher Scientific, Davenport IL) and the images were acquired with FIJI Imager Mini4000. 9. Data Analysis


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Mass spectra were processed using Proteome Discoverer (version 1.4.0.288, Thermo Scientific). Raw files were searched in Proteome Discover against the UniProt H. sapiens complete database using the Sequest HT algorithm. Trypsin was selected as the enzyme with two missed cleavages allowed. Sequest HT was searched with a parent ion tolerance of 50 ppm and a fragment ion mass tolerance of 0.02 Da. Peptide spectral matches (PSMs) were validated based on q-values to 1% FDR (false discovery rate) using percolator. Quantitation was performed in Proteome Discoverer with a reporter ion integration tolerance of 20 ppm for the most confident centroid. Only the PSMs that contained all reporter ion channels were considered, and protein quantitative ratios were determined using a minimum of one unique quantified peptide. Reporter ion ratio values for protein groups were exported to Excel workbook and corrections were performed followed by the Student t-test, which was performed with biological triplicates. The grand average hydrophobicity (GRAVY) values were calculated by the GRAVY calculator (http://www.gravy-calculator.de/). 9.1 Labeling efficiency Static modifications consisted of carbamidomethylation of cysteine residues (+57.0215 Da). Dynamic modifications consisted of isobaric labels on peptide N-termini, lysine residues (103.0833 Da for DiAla, 131.1146 Da for DiVal and 145.1303 for DiLeu) and oxidation of methionine residues (+15.9949 Da). 9.2 HEK and MOVAS protein identification and quantitation Static modifications consisted of carbamidomethylation of cysteine residues (+57.0215 Da), isobaric labels on peptide N-termini and lysine residues. Dynamic modifications was set to be oxidation of methionine residues (+15.9949 Da). 9.3 GO-term enrichment analysis Gene ontology (GO) enrichment analysis of the differentially expressed proteins by both tags was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.7

26

. Gene groups with enrichment scores ≥1.3, which is similar to P

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