Addiction to MTH1 Protein Results in Intense Expression in Human Breast Cancer Tissue As Measured by Liquid Chromatography-Isoto

Home , Isotope dilution

Addiction to MTH1 protein results in intense expression in human breast

cancer tissue as measured by liquid chromatography-isotope-dilution

tandem mass spectrometry

Erdem Coskuna,b, Pawel Jarugaa, Ann-Sofie Jemthc, Olga Losevac, Leona D.

Scanlana, Alessandro Tonad, Mark S. Lowenthala, Thomas Helledayc, Miral

Dizdaroglua,*

a Biomolecular Measurement Division, National Institute of Standards and Technology,

Gaithersburg, MD, 20899, USA

b Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey

c Science for Life Laboratory, Division of Translational Medicine and Chemical Biology,

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm,

Sweden

d Biosystems and Biomaterials Division, National Institute of Standards and Technology,

Gaithersburg, MD, 20899, USA

* Corresponding author. Tel.: +1-301-975-2581; Fax: +1-301-975-8505; E-mail address:

[email protected]

ABSTRACT

MTH1 protein sanitizes the nucleotide pool so that oxidized 2'-deoxynucleoside triphosphates (dNTPs) cannot be used in DNA replication. Cancer cells require MTH1 to avoid incorporation of oxidized dNTPs into DNA that results in mutations and cell death.

Inhibition of MTH1 eradicates cancer, validating MTH1 as an anticancer target. By overexpressing MTH1, cancer cells may mediate cancer growth and resist therapy. To date, there is unreliable evidence suggesting that MTH1 is increased in cancer cells, and available methods to measure MTH1 levels are indirect and semi-quantitative. Accurate measurement of MTH1 in disease-free tissues and malignant tumors of patients may be essential for determining if the protein is truly upregulated in cancers, and for the development and use of

MTH1 inhibitors in cancer therapy. Here, we present a novel approach involving liquid chromatography–isotope-dilution tandem mass spectrometry to positively identify and

accurately quantify MTH1 in human tissues. We produced full length 15N-labeled MTH1 and used it as an internal standard for the measurements. Following trypsin digestion, seven tryptic peptides of both MTH1 and 15N-MTH1 were identified by their full scan and product ion spectra. These peptides provided a statistically significant protein score that would unequivocally identify MTH1. Next, we identified and quantified MTH1 in human disease-free

breast tissues and malignant breast tumors, and in four human cultured cell lines, three of which were cancer cells. Extreme expression of MTH1 in malignant breast tumors was observed, suggesting that cancer cells are addicted to MTH1 for their survival. The approach described is expected to be applicable to the measurement of MTH1 levels in malignant

tumors vs. surrounding disease-free tissues in cancer patients. This attribute may help develop novel treatment strategies and MTH1 inhibitors as potential drugs, and guide

therapies.

Keywords:

MTH1 protein isotope-dilution mass spectrometry stable isotope-labeled MTH1 extreme expression nucleotide pool

Abbreviations: dNTPs, 2'-deoxynucleoside triphosphates; LC-MS/MS with isotope-dilution, liquid chromatography–isotope-dilution tandem mass spectrometry; hMTH1, human MTH1;

15N-hMTH1, full length 15N-labeled human MTH1 protein; QToF LC/MS, liquid chromatography-QToF mass spectrometry; TIC, total-ion-current; MH+, protonated molecular

ion; (M+2H)2+, doubly protonated (charged) molecular ion; SRM, selected-reaction

monitoring; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MCF-

10A cells, mammary gland epithelial cells; MCF-7 cells, mammary gland epithelial

adenocarcinoma cells; HeLa cells, cervix epithelial adenocarcinoma cells; HepG2 cells,

hepatocellular carcinoma cells; m/z, mass-to-charge; Th, thomson (the m/z unit).

1. Introduction

In aerobic organisms, intracellular metabolism and exogenous sources such as ionizing

radiation, carcinogenic compounds, etc. generate reactive species including free radicals

derived from either oxygen or nitrogen [1]. Oxidative stress thereby generated may lead to

increased genetic instability, proliferation, cell death, apoptosis and onset of inflammation,

which is a hallmark of cancer [1-4]. Reactive species are involved in carcinogenesis by

damaging DNA and by modulating certain cellular pathways [1, 3]. Among free radicals, the

hydroxyl radical reacts with DNA constituents at or near diffusion-controlled rates [5],

generating oxidatively induced DNA damage with numerous modifications (reviewed in [6,

7]).

Oxidatively induced DNA damage is repaired in vivo by various mechanisms involving

numerous DNA repair proteins [8]. DNA damage that escapes repair before replication may

lead to mutagenesis, which is a fundamental part of the molecular basis of all cancers [8-10].

Oxidatively induced damage also leads to the modification of 2'-deoxynucleoside

triphosphates (dNTPs) in the cellular nucleotide pool. MutT protein in E.coli and its homologs

in mammals (MTH1 protein) catalyze the hydrolysis of oxidized dNTPs into monophosphates

[11-20], preventing their incorporation into DNA by DNA polymerases during replication that

may lead to mutations and cell death. 8-Hydroxy-2'-deoxyguanosine triphosphate has been

first identified to be the substrate of MutT and MTH1 proteins [11, 19]. Subsequently, human

MTH1 (hMTH1) has been also shown to hydrolyze 2-hydroxy-2'-deoxyadenosine triphosphate and 8-hydroxy-2'-deoxyadenosine triphosphate [21, 22]. A number of RAS- expressing human cancers such as lung cancer and renal carcinoma have been shown to overexpress MTH1 [23-25]. Suppression of the mutator phenotype in mismatch repair- defective colorectal cancer cells has been achieved by overexpressing MTH1 [26]. Reversal of RAS-induced senescence by reducing the level of DNA damage has also been demonstrated [27]. MTH1 overexpression promoted longevity, reduced anxiety [28], and protected from neurodegeneration [29]. Compounds selectively killing RAS-expressing cells

have been found to work through targeting MTH1 [30]. MTH1 appears to be a non-essential protein in normal cells by the fact that no significant difference was observed in the survival rate of wild type and mth1─/─ mice, although more tumors were found in aged mth1─/─ mice

than in wild type mice [18]. On the other hand, cancer cells have been shown to require

MTH1 for efficient survival by preventing the incorporation of modified dNTPs that would

result in DNA damage and cell death, suggesting that this protein may be targeted as an

anticancer therapeutic approach [30, 31]. Thus, this work validated MTH1 as an anticancer

target in vivo and showed that the inhibition of MTH1 by small molecules as first-in-class

Nudix hydrolase family inhibitors eradicates cancer in patient-derived mouse xenografts.

This clearly points to the predictive and prognostic value of hMTH1 expression in human

cancers. Despite the recent progress on its importance in cancer development and therapy,

the positive identification and accurate quantification of hMTH1 in tissues has been lagging.

In general, expression levels of hMTH1 and other proteins have been estimated by semi-

quantitative Western blot methods. Here, we developed a methodology to positively identify

and accurately measure hMTH1 in human tissues using liquid chromatography-tandem

mass spectrometry (LC-MS/MS) with isotope-dilution. We measured levels of hMTH1 in

human normal and malignant breast tissues, and in four human cultured cell lines.

2. Materials and methods

2.1. Materials

Trypsin (Proteomics Grade), acetonitrile (HPLC-grade), acetonitrile plus 0.1 % formic

acid (HPLC-grade), water (HPLC-grade), water plus 0.1 % formic acid (HPLC-grade) and

trifluoroacetic acid (TFA) (HPLC grade, ≥ 99%) were purchased from Sigma (St. Louis, MO).

Water purified through a Milli-Q system (Millipore, Bedford, MA) was used for other

15 applications. N-NH4Cl was purchased from Cambridge Isotope Laboratories (Andover, MA).

Protein lysates, each of which had 150 µg of protein, were purchased from OriGene

(Rockville, MD). 5

2.2. Expression and purification of hMTH1 and 15N-labeled hMTH1

The hMTH1 p18 isoform was expressed and purified as described [32]. This is the major isoform of hMTH1 in cells. 15N-labeled hMTH1 (15N-hMTH1) was expressed from pET28a- hMTH1 in E.coli BL21(DE3) grown in a minimal medium (6 g Na2HPO4, 3 g KH2PO4, 0.5 g

15 NaCl, 5 g glucose, 120.4 mg MgSO4 per liter) containing 1 g N-NH4Cl. The production was induced by addition of 200 µmol/L isopropyl β-D-1-thiogalactopyranoside (Sigma, St. Louis,

MO). Cells were harvested by centrifugation after 16 h at 20 °C and the obtained pellet was dissolved in BugBuster protein extraction reagent (Millipore, Billerica, MA) supplemented

with complete protease inhibitor cocktail (Roche, Indianapolis, IN) and benzonase nuclease

(Novagen, Denmark). The resulting suspension was centrifuged and the cleared lysate was

subjected to chromatography on Ni-Sepharose column HisTrap HP (GE Healthcare, Bio-

Science, Uppsala, Sweden). Bound proteins were eluted using a linear gradient of imidazole

(25 mmol/L – 500 mmol/L) and analyzed by sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE). Fractions containing 15N-hMTH1 were after dialysis loaded onto a MonoQ anion-exchange column (GE Healthcare, Bio-Science, Uppsala, Sweden) and

eluted with a linear gradient of NaCl. Eluted fractions were analyzed using sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining.

Fractions containing pure 15N-hMTH1 protein were pooled and stored in 20 mmol/L HEPES,

225 mmol/L NaCl, 10 % glycerol and 1 mmol/L Tris(2-carboxyethyl)phosphine hydrochloride

(Sigma, St.Louis, MO) at ‒80 °C. High purity of the hMTH1 and 15N-hMTH1 preparations was confirmed using SDS-PAGE followed by Coomassie staining.

2.3. Molecular mass measurement of hMTH1 and 15N-hMTH1 by liquid chromatography-

QToF mass spectrometry

Intact masses of hMTH1 and 15N-hMTH1 were determined using a high-resolution

Agilent 6550 iFunnel liquid chromatography-QToF mass spectrometry (LC-QToF MS) system (Santa Clara, CA). An Agilent 1260 Infinity LC system was used to load ≈ 100 ng of

each sample at 7 µL/min onto an Atlantis dC18 nanoAcquity column (3 µm particles, 0.3 mm

x 150 mm) (Waters , Milford, MA). The Dual AJS ESI (Jet Stream electrospray ionization)

source was set to microflow. Source conditions were set as follows: gas temperature, 290° C;

drying gas, 14 L/min; nebulizer gas, 241 kPa; sheath gas temperature, 350° C; sheath gas

flow, 11 L/min; Vcap, 3500 V; nozzle voltage, 1000; fragmentor potential, 300 V. Mobile phases A and B consisted of water with 0.1 % (v/v) formic acid and acetonitrile with 0.1 %

(v/v) formic acid, respectively. Proteins were separated using a linear elution gradient from

5 % to 90 % mobile phase B over 25 min followed by a column wash and re-equilibration.

Profile data were acquired in the QToF mass spectrometer in positive polarity. Acquisition was performed as MS-only in a standard m/z range from 500 Th to 3200 Th. Collision

energy was set to 0 V, the acquisition rate was set to 2 spectra/s, and 2996 transients were

acquired per spectrum. The 6550 ToF tube was tuned in extended mass range (2 GHz) with

the slicer set to high-resolution prior to analysis. Calibration was performed to a ∆mass within

0.5 µDa/kDa using a 1:10 dilution of Agilent’s electro-spray ionization (ESI)-L tune/calibration

solution. Peak deconvolution was performed in Agilent’s MassHunter Qualitative Analysis

software (v. B.06.00) using the MaxEnt (maximum entropy) algorithm. Peaks were

deconvoluted using a mass step of 1 Da; peak S/N ≥ 30; top 90 % of peak height; ≥ six

consecutive charge states; protonated and sodiated adducts; baseline subtraction factor, 3.

2.4. Cell culture

Mammary gland epithelial cells (MCF-10A), mammary gland epithelial adenocarcinoma

cells (MCF-7), hepatocellular carcinoma cells (HepG2) and cervix epithelial adenocarcinoma

cells (HeLa) were purchased from ATCC (Manassas, VA). MCF-10A cells were grown in

Mammary Epithelium Basal Medium (MEBM) (Lonza, Walkersville, MD) supplemented with

four Mammary Epithelium Growth Medium (MEGM) SingleQuot (Lonza, Walkersville, MD)

supplements (hydrocortisone, human recombinant insulin, human recombinant epidermal

growth factor and bovine pituitary extract), 100 ng/mL cholera toxin (Sigma, St. Louis, MO)

and 1 % by volume penicillin-streptomycin (pen-strep) (Life Technologies, Grand Island, NY).

HepG2 cells were grown in Eagle’s Minimum Essential Medium (EMEM) (ATCC, Manassas,

VA), which contains non-essential amino acids, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, and 1500 mg/L sodium bicarbonate and was supplemented with 10 % by volume fetal bovine serum (FBS) (Life Technologies, Grand Island, NY) and 1 % by volume pen- strep. MCF-7 cells were grown in EMEM supplemented with 20 % by volume FBS and 0.01 mg/mL human insulin (Life Technologies, Grand Island, NY). HeLa cells were grown in

EMEM supplemented with 10 % by volume FBS. All cells were maintained in a humidified

5 % CO2 balanced-air atmosphere at 37 °C.

2.5. Protein extraction from cultured human cells

The extraction of total proteins from MCF-10A, MCF-7, HeLa and HepG2 cells was

performed using a kit, M-PER (Thermo Scientific, Rockford, IL) according to the instructions

of the manufacturer. Aliquots of 6 x 106 cells were washed twice with 1 mL PBS and then

pelleted by centrifugation at 2500 x g for 10 min. The supernatant fraction was discarded. An

aliquot of 1 mL ice-cold M-PER was added to the pellet. The mixture was pipetted up and

down to resuspend the pellet. The mixture was shaken gently for 10 min. The cell debris was

removed by centrifugation at 14000 x g for 15 min. The supernatant fraction was transferred

to a pre-chilled tube and kept at –80 °C until use. Protein concentrations in the extracts were

measured by the Bradford method [33], using the Microplate Procedure of Coomassie

(Bradford) Protein Assay Kit (#23200) (Thermo Scientific, Rockford, IL).

2.6. Separation and enrichment of MTH1 from protein extracts by HPLC

Aliquots of protein extracts from cultured cells (≈ 600 µg) were spiked with an aliquot of

15N-hMTH1 (0.04 µg) as an internal standard and mixed by vortex. Due to the large volume, the samples were ultrafiltrated using 2 mL Amicon® Ultra-2 centrifugal filter tubes with a nominal molecular mass limit of 10000 Da (Millipore, Billerica, MA). The final volume of each

extract after centrifugation for 20 min at 7500 x g was ≈ 60 µL. Protein extracts from human breast tissues (150 µg each) with a volume of ≈ 25 µL to 50 µL were spiked with an aliquot of 15N-hMTH1 (0.04 µg) and used directly. In order to isolate and enrich MTH1 prior to LC-

MS/MS analysis, protein extracts were separated by HPLC using a liquid chromatograph equipped with an automatic injector and a diode-array detector (Agilent Technologies,

Wilmington, DE), and a column specifically designed for protein separations (XBridge

Protein BEH C4 column, 4.6 mm x 250 mm, 3.5 μm) with a precolumn insert (Delta-Pak C4,

5 μm) (Waters, Milford, MA). Mobile phases A and B were water with 0.1 % TFA (v/v) and

acetonitrile with 0.1 % TFA (v/v), respectively. A gradient starting from 30 % B and linearly

increasing to 66 % B over 9 min was used. B was then increased to 90 % in 0.1 min, kept at

this level for 5 min and then decreased to 30 % to equilibrate the column for 6 min. The flow

rate was 1 mL/min. The effluents were monitored at 220 nm. Prior to separation of protein

extracts, an aliquot of hMTH1 was injected to determine its elution time period. The fractions

at the elution time period of hMTH1 (≈ 1 min) were collected, and then dried in a SpeedVac

prior to trypsin digestion.

2.7. Hydrolysis with Trypsin

An aliquot of 50 μg of hMTH1 or 15N-hMTH1 was added to 200 μL Tris-HCl buffer (30 mmol/L, pH 8.0) and incubated with 1 μg trypsin at 37 ºC for 2 h. Then, a second aliquot of 2

μg trypsin was added and incubation continued for an additional 22 h. Collected and dried fractions of protein extracts were hydrolyzed with the same procedure. Hydrolyzed samples were filtered using Nanosep® 3K Omega tubes with molecular weight cutoff of 3000 (Pall

Life Sciences, Ann Arbor, MI) for 20 min at 14000 x g in order to remove any particles and trypsin. Subsequently, filtered samples were concentrated in a SpeedVac under vacuum to ≈

50 µL prior to LC-MS/MS analysis. Tryptic hydrolysates of hMTH1 or 15N-hMTH1 were used without concentration.

2.8. Analysis by LC-MS/MS

A Thermo-Scientific Dionex Ultimate 3000 UHPLC Focused LC system and a Thermo-

Scientific Finnigan TSQ Quantum Ultra AM triple quadrupole MS/MS system with an installed heated electrospray-ionization (HESI) source were used. Experimental conditions for the automated mass calibration and operating parameters of the MS/MS system in the positive ion mode were as previously described [34], except for sheath gas (nitrogen) pressure and scan width being 60 (arbitrary units) and m/z 2, respectively. A Zorbax Extend-

C-18, Rapid Resolution HT column (2.1 mm x 100 mm, 1.8 μm particle size) (Agilent

Technologies, Wilmington, DE) with an attached Agilent Eclipse XDB-C8 guard column (2.1

mm x 12.5 mm, 5 μm particle size) was used for separations. The autosampler and column

temperature were kept at 5 ºC and 40 ºC, respectively. Mobile phase A was water plus 2 %

acetonitrile and 0.1 % formic acid (v/v). Mobile phase B consisted of acetonitrile plus 0.1 %

formic acid (v/v). The flow rate was 0.3 mL/min. An aliquot (2 µL; 0.5 µg of protein) of the

tryptic hydrolysate of hMTH1 or 15N-hMTH1, or 50 μL of tryptic hydrolysates of collected protein extracts were injected onto the LC column. A gradient starting from 1 % B and linearly increasing to 41 % B in 20 min was used. Afterwards, B was increased to 90 % in

0.1 min and kept at this level for 6 min and then decreased to 1 % to equilibrate the column for 10 min.

2.9. Statistical analysis

The statistical analysis of the data was performed using the GraphPad Prism 6 software

(La Jolla, CA, USA) and unpaired, two-tailed nonparametric Mann Whitney test with

Gaussian approximation and confidence level of 99 %.

3. Results

3.1. Production and characterization of hMTH1 and full length 15N-labeled hMTH1

We overexpressed and purified His-tagged hMTH1 and 15N-hMTH1 from E.coli. The use of 15N-hMTH1 as an internal standard is critical for accurate measurements of protein levels.

SDS-PAGE analysis of both proteins showed two bands with identical migration time, indicating high purity (Suppl.-Fig. 1A). Using HPLC, we also determined the purity and

elution behavior of each protein. One single peak was observed for each protein with

essentially identical retention times (Suppl.-Fig. 1B). We next used a high-resolution QToF

LC/MS system to measure the intact molecular masses of hMTH1 and 15N-hMTH1, and the isotopic purity of the latter. Overlapping charge-state envelopes for each protein were separately deconvoluted using MassHunter’s MaxEnt algorithm (Suppl.-Fig. 2A). Several peaks were observed for each protein representing different forms, due to N-terminal

methionine truncation ([MH]+ ‒ Met), or due to other chemical processing ([MNa]+ ‒ Met and

[MH]+ + glucuronic acid ions). Suppl.-Fig. 2B shows overlay of the overlapping charge-state

envelopes for the heterogeneous mixtures of hMTH1-related protein forms observed from

the high-resolution MS1 mass spectral data acquisition of hMTH1 and 15N-hMTH1. The inset demonstrates an enlarged view for three different charge states of each protein. The observed presence of two or four major protein forms for hMTH1 and 15N-hMTH1, respectively, was demonstrated by the largest peaks with the potential contribution from

minor protein forms also being observed. The charge-state envelopes were deconvoluted by

software algorithms into intact molecular masses for each protein (Suppl.-Fig. 2B). The

calculated average molecular masses (MH+) of His-tagged hMTH1 and 15N-hMTH1 amount to 20115.600 Da and 20361.965 Da, respectively, assuming that all the N-atoms are labeled in 15N-hMTH1. The measured average molecular masses (MH+) of hMTH1 and 15N-hMTH1 were 19984.13 Da and 20216.20 Da, respectively, indicating that an amino acid was missing

from both molecules. It is well known that the N-terminal Met is excised from eukaryotic

proteins by the actions of peptide deformylase and methionine aminopeptidase as an

essential process, when the penultimate residue is small and uncharged such as Gly next to

Met in His-tagged hMTH1 [35]. Without the N-terminal Met, the calculated average molecular

masses of hMTH1 and 15N-hMTH1 amounted to 19984.404 Da and 20229.776 Da, respectively. The measured average molecular mass of hMTH1, 19984.13 Da differed from that of the theoretical value by 0.27 Da, approximately 1 µDa/kDa, well within the mass accuracy error of the QToF mass spectrometer over the measured mass range. In the case of 15N-hMTH1, the mass difference between the theoretical and observed average molecular masses was 13.6 Da. This is 5.5 % of the 246 N-atoms in 15N-hMTH1, suggesting that at least 94.5 % of these atoms were replaced by 15N-atoms. These data confirmed the identity of both hMTH1 and 15N-hMTH1, and suggested an almost complete labeling of 15N-hMTH1, enabling its proper use as an isotopic equivalent analog for the analysis of hMTH1 by LC-

MS/MS.

The amount of 15N-hMTH1 in aqueous solution was measured using the Bradford method [33], and also by absorption spectrophotometry using the extinction coefficient of

27960 M‒1 cm‒1 at 280 nm, which was calculated using ExPASy ProtParam program

(http://web.expasy.org/cgi-bin/protparam/protparam) [36]. The absorption spectrum of 15N-

hMTH1 is shown in Suppl. Fig. 3. The measured amounts of 15N-hMTH1 by both methods agreed well with each other, amounting to 1.57 ± 0.06 μg/μL (n = 6). The concentration of hMTH1 was 1.29 ± 0.06 μg/μL (n = 6). The uncertainties are standard deviations. The ratio of these two values was in excellent agreement with the ratio of the peak areas of both proteins measured by HPLC as shown in Suppl. Fig. 1B.

3.2. Separation and identification of tryptic peptides of hMTH1 and 15N-hMTH1

We hydrolyzed hMTH1 and 15N-hMTH1 with trypsin and used LC-MS/MS to separate and identify resulting tryptic peptides by their full-scan and product ion mass spectra.

According to the “Peptide Cutter” program (http://web.expasy.org/peptide_cutter/), trypsin

cleaves 18 peptide bonds in His-tagged hMTH1, resulting in 2 arginines, 3 lysines and 14

peptides containing 5 to 36 amino acids. Fig. 1A illustrates the total-ion-current (TIC) profile of the tryptic hydrolysate of hMTH1. Seven peptides that matched the theoretical tryptic peptides of hMTH1 were identified based on their full-scan mass spectra. Suppl. Fig. 4

shows the sequence of His-tagged hMTH1 with the expected and identified tryptic peptides.

The tryptic hydrolysate of 15N-hMTH1 yielded an essentially identical TIC profile (Fig. 1B).

Table 1 shows the identities of the tryptic peptides of hMTH1 and 15N-hMTH1, and the monoisotopic masses of their protonated molecular ions (MH+) and doubly protonated

(charged) molecular ions [(M+2H)2+]. Typical (M+2H)2+ and MH+ ions were observed in the

mass spectra of seven tryptic peptides of both proteins. As an example, Fig. 2A shows the

mass spectrum of VQEGETIEDGAR (peak 2 in Fig. 1A) with (M+2H)2+ at m/z 652 and MH+

at m/z 1303. The mass spectrum of 15N-VQEGETIEDGAR (peak 2 in Fig. 1B) exhibited a shift of 16 Da in the mass of MH+ (m/z 1319) (Fig. 2B), consistent with the sixteen 15N atoms in the molecule. Accordingly, (M+2H)2+ appeared at m/z 660. Another example is shown in

Suppl.-Fig. 5. The mass spectrum of VLLGMK (peak 5 in Fig. 1A) contained (M+2H)2+ at m/z

330 and MH+ at m/z 660 (Suppl.-Fig. 3A). As shown in Suppl.-Fig. 3B, the masses of these

ions were shifted to m/z 334 and m/z 667, respectively, in the mass spectrum of 15N-

VLLGMK (peak 5 in Fig. 1B), reflecting the presence of seven 15N-atoms in the molecule.

These spectra and those of other 15N-labeled tryptic peptides showed no unlabeled material in these peptides. The observed masses of MH+ and (M+2H)2+ ions matched the theoretical

masses shown in Table 1.

The identified tryptic peptides cover 41.7 % of the major isoform p18 of hMTH1 with 156

amino acids and a wide range in the sequence of the protein from Val-18 to Arg-151 (Suppl.

Fig. 4). With the use of the “Mascot” search engine (http://www.matrixscience.com; the

database SwissProt 2015) and the taxonomy Homo sapiens (20200 sequences), identified

tryptic peptides yielded a top protein score of 106; protein scores greater than 70 are

considered significant (p < 0.05) for positive identification. Furthermore, using the SwissProt

database (http://prospector.ucsf.edu/prospector/cgi-bin/mssearch.cgi) and three different

combinations of four out of seven tryptic peptides, a 100 % match with hMTH1 was obtained, meaning that the simultaneous measurement of just four tryptic peptides is sufficient to positively identify and quantify hMTH1.

3.3. Product ion spectra of the tryptic peptides of hMTH1 and 15N-hMTH1

Using NIST Mass and Fragment Calculator, we calculated the masses of typical y-series ions as the product ions [37], which are expected to result from the fragmentation of the tryptic peptides of hMTH1 and 15N-hMTH1. The calculated masses of y-ions are given in

Suppl.-Table 1. Product ion mass spectra were recorded, using previously determined optimal collision energies for (M+2H)2+ ions of peptides with similar masses [37, 38]. These

spectra were dominated by y-ions. The masses of observed y-ions matched the theoretical

values given in Suppl.-Table 1. As an example, Fig. 3A and B illustrate the product ion

spectra of VQEGETIEDGAR and 15N-VQEGETIEDGAR, respectively, which exhibited the y- ion series from the y3-ion to the y11-ion. Another example is shown in Suppl.-Fig. 6A and B.

15 FQGQDTILDYTLR and N-FQGQDTILDYTLR yielded the y-ion series from the y3-ion to the

15 y12-ion. The product ion mass spectra of all seven N-labeled tryptic peptides showed no unlabeled material in these peptides.

Although previously determined optimal collision energies of peptides with similar masses were used to obtain the product ion spectra of the tryptic peptides, we also determined these energies experimentally. Selected-reaction monitoring (SRM) was used to record the transitions from (M+2H)2+ to the most intense ion in the product ion spectra.

Suppl.-Fig. 7A shows the fragmentation intensity versus collision energy. Values at the

maximum of each graph were used as the optimal collision energy for the rest of this study.

A plot of the optimal collision energies versus the m/z values of (M+2H)2+ ions yielded a

linear relationship (Suppl.-Fig. 7B), which is essentially identical to those previously obtained

for other peptides with similar masses [37-39].

3.4. Selected-reaction monitoring

Selected-reaction monitoring was used to analyze the tryptic hydrolysate of a mixture of hMTH1 and 15N-hMTH1. (M+2H)2+ ions were chosen as the precursor ions for transitions.

Ion-current profiles of the mass transitions of tryptic peptides of hMTH1 and 15N-hMTH1 exhibited excellent peak shapes and a base-line separation between the peptides with co- elution of each peptide and its respective 15N-labeled analog. Suppl.-Fig. 8 illustrates ion- current profiles of mass transitions of seven tryptic peptides of hMTH1 and 15N-hMTH1. The analytical sensitivity of the instrument was determined using SRM and a number of tryptic peptides of hMTH1 and the transition from (M+2H)2+ to the most intense ion in product ion

spectra. The limit of detection (LOD) amounted to ≈ 10 fmol, with a signal-to-noise ratio (S/N)

of at least 3. The limit of quantification (LOQ) was ≈ 50 fmol of a target peptide with an S/N

of 10.

3.5. Identification and quantification of MTH1 in four human cell lines

Using the developed methodology, we first attempted to identify and quantify MTH1 in

cultured human MCF-10A, MCF-7, HeLa and HepG2 cell lines. Total protein content was

extracted from ten independent aliquots of each cell line. Protein concentration of each

extract was measured as described above. Aliquots of the extracts (600 µg protein) were

spiked with an aliquot of 15N-hMTH1 (0.04 µg) and then separated by HPLC to enrich hMTH1 for analysis by LC-MS/MS. As examples, Suppl.-Fig. 9 illustrates superimposed

elution profiles of protein extracts from each cell line, along with the superimposed elution

profile of hMTH1, which was separately obtained under identical conditions. Fractions of

protein extracts corresponding to the elution period of hMTH1 (≈ 1 min, indicated with dotted

lines in Suppl.-Fig. 9) were collected. Fractions were lyophilized, hydrolyzed with trypsin and

analyzed by LC-MS/MS. Using the procedures outlined above, the typical mass transitions of

the seven identified peptides of hMTH1 and their 15N-labeled analogs were monitored by

SRM to identify and quantify MTH1 in ten independently prepared batches of each cell line.

As examples of the identification, Fig. 4A and B, and Suppl.-Fig. 10A and B illustrate mass

transitions of four tryptic peptides of hMTH1 and their 15N-labeled analogs obtained with

each cell line. The signals of the mass transitions of four peptides of hMTH1 and 15N-hMTH1 were observed at the expected retention times, unequivocally identifying hMTH1 in these cell lines. The level of MTH1 was calculated using the measured signals of mass transitions of these peptides and their 15N-labeled analogs, and the known amount of the internal standard and the protein amount. The means of independent measurements of each peptide were combined to calculate the mean level of hMTH1 and the uncertainty of the measurements.

Fig. 5 illustrates the levels of hMTH1 in each cell line. MCF-7 cells exhibited an almost 4-fold

greater level of hMTH1 than MCF-10A cells with p < 0.0001 and a confidence level of 99 %.

HeLa cells also had a high hMTH1expression, close to that of MCF-7 cells. However, HepG2

cells exhibited quite a low hMTH1expression compared to the other three cell lines. The

levels of hMTH1 in MCF-10A, MCF-7 and HepG2 cells were ≈ 30-fold, 20-fold and 100-fold,

respectively, lower than those of hAPE1 protein, which had been also measured by LC-

MS/MS with isotope-dilution [39]. This fact indicates the ability of our methodology to

measure very low amounts of proteins in cells.

3.6. Identification and quantification of hMTH1 in human disease-free breast tissues and

malignant breast tumors

Using the same strategy above, we next attempted to measure hMTH1 levels in human

disease-free breast tissues and malignant breast tumors to test the suitability of the

developed methodology for the measurement of hMTH1 in human tissues. We used

commercially available protein extracts isolated from disease-free breast tissues of eight

individuals and from malignant breast tumors of twenty breast cancer patients. No more than

eight protein extracts of disease-free breast tissues were available to purchase. Human

tissue samples other than those commercially available could not be used at the present

time because of the difficulties in obtaining material transfer agreements with other

institutions. Suppl.-Table 2 shows the list of control individuals and patients with their characteristics. Aliquots of 150 µg of protein extracts were spiked with an aliquot of 15N- hMTH1 (0.04 µg) and then separated by HPLC to enrich hMTH1. As examples, Suppl.-Fig.

11 illustrates the superimposed elution profiles of protein extracts from a disease-free individual and a cancer patient along with the superimposed elution profile of hMTH1.

Fractions corresponding to the elution period of hMTH1 were collected, dried and hydrolyzed with trypsin, and then analyzed by LC-MS/MS. Fig. 6A and B illustrates mass transitions of four tryptic peptides of hMTH1 and their 15N-labeled analogs obtained with protein extracts from a disease-free breast tissue and a malignant breast tumor, respectively. The signals of four tryptic peptides of hMTH1 and 15N-hMTH1were observed at the expected retention times. The level of MTH1 was calculated as described above for the quantification hMTH1 in cultured cells. These measurements unequivocally identified and quantified hMTH1 in all human breast tissues tested here. The scattered data plot in Fig. 7 shows the measured levels of hMTH1 in individual breast tissues. The numbers identify the individual cancer

patients shown in Suppl. Table 2. In most cases, malignant tumors exhibited extreme

expression of hMTH1 when compared to disease-free tissues. In a few cases of malignant

tissues, however, the hMTH1 levels were similar to those in disease-free tissues. Overall,

the statistical difference between the two types of tissues was highly significant with p <

0.0001 and a confidence level of 99 %.

4. Discussion

Recent studies showed that hMTH1 is an excellent target for inhibition in cancer therapy

[30, 31]. Since hMTH1 is overexpressed in many cancers, and its activity markedly

increased [23-25, 30, 31], the level of its expression may affect cancer phenotype and

response of various tumors to conventional treatments and to future combination therapies

using inhibitors of hMTH1 as drugs. Many treatments target the genetic defects found in

cancers; however, the diversity and complexity of mutational processes underlying the

development of cancer may have potential implications for therapy and limit targeting genetic defects in future personalized therapies [40]. The efficacy of therapeutic agents may be influenced by DNA repair capacity, which is likely to be increased in tumors by overexpression of DNA repair proteins [41-46]. Similarly, the efficacy of treatments targeting hMTH1 may depend on its expression levels in normal tissues and malignant tumors, which may depend on the type of cancer, affected organ and patient condition among other factors.

This means that knowledge of hMTH1 expression levels in malignant tumors as well as in surrounding disease-free tissues in a patient will be essential for achieving optimal therapeutic results. Such knowledge may also help evaluate MTH1 as a predictive and prognostic biomarker in cancer, and develop personalized treatment strategies that may involve inhibitors of hMTH1 as cancer therapy drugs.

Thus far, methods such as Western blotting have been used to estimate hMTH1 levels and other proteins in vivo. These methods separate proteins by SDS-PAGE and use antibodies specific to a target protein, completely depending on a reliable and specific antibody resource. No mass spectrometric evidence, thus no positive identification is provided. Without the use of internal standards, the estimation of protein amounts is based on the stained area. The molecular mass of the target protein is estimated only by checking against a molecular mass ladder of proteins. Moreover, antibodies may potentially exhibit some off-target binding, leading to false identification and/or quantification of the target protein.

We developed a novel approach for the positive identification and absolute quantification of hMTH1 in human tissues using LC-MS/MS with isotope dilution. We generated full length

15N-hMTH1 and used it as an internal standard. A stable isotope-labeled internal standard, which possesses identical chemical and physical properties to the target protein, can be added into protein extracts at the earliest step of sample preparation. This attribute is required for enrichment of target proteins by HPLC or by any other procedure, and compensates for eventual losses during all stages of analysis. Furthermore, the

measurement bias due to trypsin hydrolysis, which can often be inefficient depending on the measured protein, can be avoided.

First, we achieved the positive identification and absolute quantification of hMTH1 in four cultured human cell lines. We used a large number of independently prepared replicates (n =

10) of each cell line to ascertain the accuracy of measurements. We found statistically significant differences between hMTH1 levels. Two of them MCF-10A and MCF-7 are mammary gland epithelial cells, with the latter being adenocarcinoma cells. The measurement of hMTH1 in these cells under identical experimental conditions permitted the comparison of its levels in normal cells vs. adenocarcinoma cells of the same origin. MCF-7

cells exhibited an almost 4-fold greater level of hMTH1 than MCF-10A cells with highly

statistical significance (p < 0.0001) and confidence level (99 %). Among the cell lines, MCF-7

cells had the highest level of hMTH1 with the lowest level in HepG2 cells. There were

significant differences between MCF-7 cells and HeLa cells (p < 0.0004), between MCF-7

cells and HepG2 cells (p < 0.0001), and between HeLa cells and HepG2 cells (p < 0.0001).

The MTH1 level in HepG2 cells was even lower than that in normal MCF-10A cells (p <

0.0001). Confidence level was 99 % in all cases. These results also show the ability of the

developed methodology to detect and quantify differences in hMTH1 amounts between cell

lines.

Encouraged by the results obtained with cultured cell lines, we tested the ability of the

developed methodology to measure hMTH1 expression levels in protein extracts isolated

from human disease-free breast tissues and malignant breast tumors. We achieved the

identification and quantification of hMTH1 in these human tissues as well. Commercially

available human protein extracts were used only because of the difficulties in obtaining

material transfer agreements with other institutions. When compared to disease-free breast

tissues, most of the malignant breast tumors exhibited extreme expression of hMTH1.

Among twenty samples, a few had hMTH1 levels similar to those observed with disease-free

breast tissues. hMTH1 levels in disease-free breast tissues were quite similar to one another. 19

Taken together, the difference between hMTH1 levels in disease-free tissues and malignant tumors was highly significant, suggesting that cancer cells are addicted to MTH1 for survival.

This observation is on a par with the highly significant expression of hMTH1 in MCF-7 adenocarcinoma cells when compared with MCF-10A normal cells of the same origin. Our findings are in agreement with those showing that MTH1 is overexpressed in many cancers

[23, 24, 30, 31]. Furthermore, MTH1 expression has been found to be strongly correlated with upregulated RAS, as cancers with high prevalence of ras mutations such as lung and colon cancers have been found to express greater levels of MTH1 than other unrelated cancers [30, 31]. However, all cancers are not equal, and expression of MTH1 strongly differs between cancers [30, 31]. Our results on three cultured cancer cells are also in agreement with previously observed different expression levels of MTH1 in cancers. Much lower amounts of hMTH1 were observed in tissues than in cultured cells. This may indicate the effect of cell culturing on expression levels of proteins and reflect the differences

between cell culture and mammalian tissues. It is also well known that cell cultures are only

one cell type, whereas tissues consist of mixtures of cell types. A similar difference in

expression levels of APE1 protein was observed when its amount was measured by LC/MS-

MS with isotope dilution in human cultured cells and mouse liver [39]. Again, the

identification and quantification of hMTH1 in human tissues at very low levels attest to the

ability of the developed methodology to accurately measure low protein levels.

In our work, we used two human cell lines of the same origin to show significantly greater

expression level of hMTH1 in the cancer cell line. In the case of human tissues, however, we

could only use samples from different individuals. It is well known that the expression levels

of a protein may differ among individuals. Nevertheless, hMTH1 levels in protein extracts

from eight disease-free individuals were quite similar. A few of malignant breast tissues

exhibited expression levels similar to those in controls. Due to limitation to obtain human

samples, we could not compare hMTH1 levels in malignant tumors with those in surrounding

disease-free tissues in the same breast cancer patient. This type of a comparison, of course,

would be desirable in future applications of our approach to clinical samples. This attribute may help develop novel treatment strategies, and guide not only the conventional therapies, but also the development and use of hMTH1 inhibitors as potential anticancer drugs.

Conflict of interest statement

The authors state that there are no conflicts of interests regarding the above-mentioned manuscript submitted for publication in DNA Repair.

Acknowledgements

This work was supported by the Knut and Alice Wallenberg Foundation (T.H.), Swedish

Research Council (T.H.), Swedish Cancer Society (T.H.), the Swedish Pain Relief

Foundation (T.H.), the Swedish Foundation for Strategic Research (T.H.), the Göran

Gustafsson Foundation and the Torsten and Ragnar Söderberg Foundation (T.H.).

Certain commercial equipment or materials are identified in this paper in order to specify

adequately the experimental procedure. Such identification does not imply recommendation

or endorsement by the National Institute of Standards and Technology, nor does it imply that

the materials or equipment identified are necessarily the best available for the purpose.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at….

Table 1. Identification of the tryptic peptides in Fig. 1 and the m/z values (Th) of the monoisotopic masses of their MH+ and (M+2H)2+ ions.

unlabeled 15N-labeled

peak tryptic peptide MH+ (M+2H)2+ MH+ (M+2H)2+

1 GFGAGR 564.29 282.65 573.26 287.14

2 VQEGETIEDGAR 1303.61 652.31 1319.57 660.29

3 FHGYFK 798.39 399.70 807.36 404.19

4 WNGFGGK 765.37 383.19 775.34 388.17

5 VLLGMK 660.41 330.71 667.39 334.20

6 ELQEESGLTVDALHK 1668.84 834.93 1687.78 844.40

7 FQGQDTILDYTLR 1569.79 785.40 1587.74 794.37

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Figure Captions

Fig. 1. TIC profiles of the tryptic hydrolysates of hMTH1 (A) and 15N-hMTH1 (B). The

identities of the peptides are given in Table 1.

Fig. 2. Full-scan mass spectra of VQEGETIEDGAR (A) (represented by peak 2 in Fig. 1A)

and 15N-VQEGETIEDGAR (B) (represented by peak 2 in Fig. 1B).

Fig. 3. Product ion spectra of VQEGETIEDGAR (A) (represented by peak 2 in Fig. 1A) and

15N-VQEGETIEDGAR (B) (represented by peak 2 in Fig. 1B). The (M+2H)2+ ions m/z 652.31

and m/z 660.29 (Table 1), respectively, were used as the precursor ions.

Fig. 4. Ion-current profiles of mass transitions of four tryptic peptides of hMTH1 and 15N- hMTH1 obtained using the tryptic hydrolysate of a protein fraction, which was collected during separation by HPLC of protein extracts of MCF-10A (A) and MCF-7 (B). Protein extracts were spiked with an aliquot of 15N-hMTH1 prior to separation. Peptides and monitored transitions are also shown.

Fig. 5. Levels of hMTH1 in MCF-10A, MCF-7, HeLa and HepG2 cells. Ten independently prepared replicates of each protein extract were used for each data point. The uncertainties are standard deviations. Statistical significance: MCF-10A vs. MCF-7, p <0.0001; MCF-7 vs.

HeLa, p <0.0004; MCF-7 or HeLa vs. HepG2, p <0.0001. In all cases, the confidence level was 99 %.

Fig. 6. Ion-current profiles of mass transitions of four tryptic peptides of hMTH1 and 15N- hMTH1 obtained using the tryptic hydrolysates of protein extracts from a disease-free breast tissue (A) and a malignant breast tumor (B).

Fig. 7. Levels of hMTH1 in disease-free breast tissues and in malignant breast tumors. The details of the samples are given in Suppl.-Table 2. The uncertainties are standard deviations.

Median with interquartile range is shown. Statistical significance: normal vs. cancer, p

<0.0001 with a confidence level 99 %. The numbers indicate the individual cancer patients shown in Suppl. Table 2.

5 A 5.4e7 10.15 6 4.8e7 10.78

4.2e7

3.6e7 3 4 7 3.0e7 8.14 9.17 14.12

ntensity 2 i 7.52 2.4e7 1 6.94 4.60 1.8e7

1.2e7

0.6e7

3 4 5 6 7 8 9 10 11 12 13 14 15 16 time (min)

5 6 B 10.75 8e7 10.13

7e7

6e7

5e7 3 4 8.14

ntensity 4e7 9.17 i 1 2 7 4.54 6.91 14.12 7.54 3e7

2e7

1e7

3 4 5 6 7 8 9 10 11 12 13 14 15 16 time (min)

Fig. 1 100 652 (M+2H)2+ A 90

70 VQEGETIEDGAR (%)

50 bundance a 40

30 elative elative r MH+ 20 1303 10

0 600 700 800 900 1000 1100 1200 1300 1400 mass-to-charge (m/z)

100 660 (M+2H)2+ B 90

80 15N- (%) 70 VQEGETIEDGAR

50 bundance a 40

elative elative 30 r

20 MH+ 1319 10

0 600 700 800 900 1000 1100 1200 1300 1400 mass-to-charge (m/z)

Fig. 2 b 2 1076.5 y A 100 10 V–Q–E–G–E–T–I–E–D–G–A–R 90

y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 80 y3 70 303.2

b2 60 227.9 y9 y7 947.6 50 761.4 abundance (%) 40 y5 547.2 30 y

elative elative 6

r y4 660.4 y8 20 418.2 y 891.0 11 10 1204.5

0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 mass-to-charge (m/z)

1089.5 y 100 b2 10 B 15 90 N-V–Q–E–G–E–T–I–E–D–G–A–R y 80 y11 y10 y9 8 y7 y6 y5 y4 y3 y2

y3 70 308.9

50 y9 b 960.2

abundance (%) 2 40 230.8 y5 y7 y4 554.9 771.9 424.9 y elative elative 30 y 8

r 6 901.7 669.2 20

y11 10 1220.0

0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 mass-to-charge (m/z)

Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Supplementary Table 1. The masses (Da) of the theoretical y-series ions of the tryptic peptides of hMTH1 and their 15N-labeled analogs identified in this work.

Peptide y1 y2 y3 y4 y5 y6 y7 y8 y9 y10 y11 y12 y13 y14

GFGAGR 175.12 232.14 303.18 360.20 507.27

15N-GFGAGR 179.11 237.13 309.16 367.18 515.24

VQEGETIEDGAR 175.12 246.16 303.18 418.21 547.25 660.33 761.38 890.42 947.44 1076.49 1204.54 15N-VQEGETIEDGAR 179.11 251.14 309.16 425.18 555.22 669.30 771.35 901.39 959.41 1089.45 1219.40 FHGYFK 147.11 294.18 457.25 514.27 651.33 15N-FHGYFK 149.11 297.17 461.23 519.25 659.30 WNGFGGK 147.11 204.13 261.16 408.22 465.25 579.29 15N-WNGFGGK 149.11 207.13 265.14 413.21 471.23 587.27

VLLGMK 147.11 278.15 335.18 448.26 561.34 15N-VLLGMK 149.11 281.14 339.16 453.24 567.33 ELQEESGLTVDALHK 147.11 284.17 397.26 468.29 583.32 682.39 783.44 896.52 953.54 1040.57 1169.62 1298.66 1426.72 1539.80 15N-ELQEESGLTVDALHK 149.11 289.16 403.24 475.27 591.30 691.36 793.40 907.49 965.51 1053.54 1183.58 1313.61 1443.67 1557.75 FQGQDTILDYTLR 175.12 288.20 389.25 552.31 667.34 780.43 893.51 994.56 1109.58 1237.64 1294.66 1422.72 15N-FQGQDTILDYTLR 179.11 293.19 395.23 559.29 675.32 789.40 903.48 1005.52 1121.55 1251.60 1309.62 1439.67

1 Supplementary Table 2. Breast tissue samples and their characteristics as listed on the website of OriGene (http://www.origene.com/tissue/tissueSearch.aspx) and corresponding numbers and levels of hMTH1 on Fig. 7.

Number Level of hMTH1 Type Stage Age Ethnicity (all females) on Fig. 7 (ng/μg protein) within normal limits N/A 32 black or African American 0.00010 within normal limits N/A 22 not reported 0.00024 within normal limits N/A 34 black or African American 0.00036 within normal limits N/A 63 not reported 0.00040 within normal limits N/A 72 white or caucasian 0.00059 within normal limits N/A 61 not reported 0.00077 within normal limits N/A 45 black or African American 0.00088 within normal limits N/A 30 black or African American 0.00108

adenocarcinoma of breast, ductal, mucinous IIIB 86 not reported 1 0.00057 adenocarcinoma of breast, ductal, metastatic IIB 71 white or caucasian 2 0.00063 adenocarcinoma of breast, lobular IIIA 49 not reported 3 0.00085 adenocarcinoma of breast, ductal IIIC 27 not reported 4 0.00135 adenocarcinoma of breast, lobular IIA 77 white or caucasian 5 0.00142 adenocarcinoma of breast, ductal IIB 44 not reported 6 0.00171 adenocarcinoma of breast, ductal IIA 46 not reported 7 0.00211 adenocarcinoma of breast, ductal IIA 57 not reported 8 0.00241 adenocarcinoma of breast, ductal IIIA 34 white or caucasian 9 0.00298 adenocarcinoma of breast, ductal IIC 50 not reported 10 0.00306 adenocarcinoma of breast, ductal I 44 white or caucasian 11 0.00310 adenocarcinoma of breast, ductal IIB 89 not reported 12 0.00338 adenocarcinoma of breast, ductal IIB 57 white or caucasian 13 0.00340 adenocarcinoma of breast, ductal I 89 not reported 14 0.00353 adenocarcinoma of breast, metastatic IV 55 not reported 15 0.00359 adenocarcinoma of breast, metastatic IV 41 white or caucasian 16 0.00426 adenocarcinoma of breast, ductal IIIC 61 not reported 17 0.00645 adenocarcinoma of breast, ductal IIIA 53 not reported 18 0.00705 adenocarcinoma of breast, ductal, metastatic IIIA 40 not reported 19 0.00995 adenocarcinoma of breast, ductal IIC 56 not reported 20 0.01223

2 B 15N-hMTH1

hMTH1

Supplementary Figure 1. A: SDS-PAGE analysis of hMTH1 and 15N-hMTH1; B: Separation of hMTH1 and 15N-hMTH1 by HPLC. The elution profiles were superimposed. A hMTH1 15 [MH]+ ─ Met N-hMTH1 19984.13 Da

[MH]+ ─ Met 20216.20 Da

[MH]+ + glucuronic acid [MNa]+ ─ Met 20162.41 Da 20239.09 Da + [MH] ─ H2O [MNa]+ ─ Met 20348.50 Da 20006.63 Da

[MNa]+ + elative abundance elative r glucoronic [MH]+ + acid glucuronic acid 20184.76 Da 20394.67 Da

molecular mass (Da)

B hMTH1

15N-hMTH1

elative abundance elative r

mass-to-charge (m/z)

Supplementary Figure 2. Measurement of the masses of hMTH1 and 15N-hMTH1 by QToF LC/MS. A: Mass spectra of hMTH1 and 15N-hMTH1; B: Deconvolution of the mass spectral data Supplementary Figure 3. Absorption spectrum of 15N-hMTH1. MGSSHHHHHHSSGLVPR GSHMGASR LYTLVLVLQPQR VLLGMK K R GFGAGR WNGFGGK VQEGETIEDGAR R ELQEESGLTVDALHK VGQIVFEFVGEPELMDVHVFCTDSIQGTPVESDEMR PCWFQLDQIPFK DMWPDDSYWFPLLLQK K K FHGYFK FQGQDTILDYTLR EVDTV

Supplementary Figure 4. Sequence of the His-tagged isoform p18 of hMTH1 with expected tryptic peptides and free amino acids. Peptides in red and underlined are the identified tryptic peptides. The sequence of the His-tag is indicated in blue. 100 330.5 (M+2H)2+ A 90

70 VLLGMK (%)

bundance bundance a 40

30 elative elative r MH+ 20 660.1

0 300 340 380 420 460 500 540 580 620 660 700 mass-to-charge (m/z)

100 334.1 (M+2H)2+ B 90

80 (%) 70 15N-VLLGMK

bundance bundance 50 a

40 elative elative r 30 MH+ 20 667.3 10

0 300 340 380 420 460 500 540 580 620 660 700 mass-to-charge (m/z) Supplementary Figure 5. Full-scan mass spectra of VLLGMK (A) (represented by peak 5 in Figure 1A) and 15N-VLLGMK (B) (represented by peak 5 in Figure 1B). b2 A y4 100 552.2 F–Q–G–Q–D–T–I–L–D–Y–T–L–R 90 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2

80 b2 1295.1 276.4 y11 70 y6 60 780.4

y5 50 667.2 y9 y8

abundance abundance (%) 1109.7 40 y7 994.7 893.5

30 y3 elative elative

r 389.2 y10 20 1238.2

10 y12 1423.1 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 mass-to-charge (m/z)

b2 B y4 559.2 100 15N-F–Q–G–Q–D–T–I–L–D–Y–T–L–R

90 y y11 y y y y y y y y y b2 12 10 9 8 7 6 5 4 3 2 279.0 1310.0 y 80 11

70 y6 y8 y 60 5 789.1 1005.8 675.2 50 y9 abundance abundance (%) y7 y 1122.0 3 903.7

40 395.2 elative elative r 30

y10 20 1252.0 10 y12 1440.1 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 Mass-to-charge (m/z) Supplementary Figure 6. Product ion spectra of FQGQDTILDYTLR (A) (represented by peak 7 in Figure 1A) and 15N-FQGQDTILDYTLR (B) (represented by peak 7 in Figure 1B). The (M+2H)2+ ions m/z 785.40 and m/z 794.37 (Table 1), respectively, were used as the precursor ions.

Supplementary Figure 8. Ion-current profiles of mass transitions of seven tryptic peptides of hMTH1 and 15N-hMTH1 obtained using a tryptic hydrolysate of a mixture of hMTH1 and 15N-hMTH1. Peptides and monitored transitions are also shown. Supplementary Figure 9. Superimposed elution profiles of protein extracts from MCF10A, MCF-7, HeLa and HepG2 cells. The elution profile of hMTH1 was also superimposed to show the retention time period where collections were made to enrich hMTH1 for subsequent analysis by LC-MS/MS. The retention time of hMTH1 is different from that in Suppl. Fig. 1B because a different HPLC elution program was used in this case. A B GFGAGR GFGAGR m/z 282.6 → m/z 360.2 m/z 282.6 → m/z 360.2

15N-GFGAGR 15N-GFGAGR m/z 287.1 → m/z 367.2 m/z 287.1 → m/z 367.2

VQEGETIEDGAR VQEGETIEDGAR m/z 652.3 → m/z 1076.5 m/z 652.3 → m/z 1076.5

15N-VQEGETIEDGAR 15N-VQEGETIEDGAR m/z 660.3 → m/z 1089.5 m/z 660.3 → m/z 1089.5

VLLGMK VLLGMK m/z 330.7 → m/z 448.3 m/z 330.7 → m/z 448.3

15N-VLLGMK 15N-VLLGMK m/z 334.2 → m/z 453.2 m/z 334.2 → m/z 453.2

FQGQDTILDYTLR FQGQDTILDYTLR m/z 785.4 → m/z 552.3 m/z 785.4 → m/z 552.3

15N-FQGQDTILDYTLR 15N-FQGQDTILDYTLR m/z 794.4 → m/z 559.3 m/z 794.4 → m/z 559.3

Supplementary Figure 10. Ion-current profiles of mass transitions of four tryptic peptides of hMTH1 and 15N-hMTH1 obtained using the tryptic hydrolysate of a protein fraction, which was collected during separation by HPLC of protein extracts of HeLa (A) and HepG2 (B) cells. Protein extracts were spiked with an aliquot of 15N-hMTH1 prior to separation. Peptides and monitored transitions are also shown. Supplementary Figure 11. Superimposed elution profiles of protein extracts from a disease-free breast tissue and a malignant breast tumor. The elution profile of hMTH1 was also superimposed to show the retention time period where collections were made to enrich hMTH1 for subsequent analysis by LC-MS/MS. The retention time of hMTH1 is different from that in Suppl. Fig. 1B because a different HPLC elution program was used in this case.