Liquid Chromatography-High Resolution Mass Spectrometry Analysis

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Liquid Chromatography-High Resolution Mass Spectrometry Analysis of Platelet Frataxin as a Protein Biomarker for the Rare Disease Friedreich’s Ataxia Lili Guo, Qingqing Wang, Liwei Weng, Lauren A. Hauser, Cassandra J. Strawser, Agostinho G. Rocha, Andrew Dancis, Clementina A Mesaros, David R. Lynch, and Ian Alexander Blair Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04590 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 23, 2017

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Analytical Chemistry

Analytical Chemistry submission 1

Liquid Chromatography-High Resolution Mass Spectrometry

2

Analysis of Platelet Frataxin as a Protein Biomarker for the Rare

3

Disease Friedreich’s Ataxia

4 §

Qingqing Wang,a,b,

§

5

Lili Guo,a,b,

Liwei Weng,a Lauren A. Hauser,b,c,d

6

Cassandra J. Strawser,b,c,d Agostinho G. Rocha,e

7

Clementina Mesaros,a,b David R. Lynch,b,c,d and Ian A. Blaira,b,*

¶

Andrew Dancis,e

8 9

a

Penn SRP Center and Center of Excellence in Environmental Toxicology Center, Department

10

of Systems Pharmacology and Translational Therapeutics Perelman School of Medicine,

11

University of Pennsylvania Philadelphia, PA 19104, United States

12

b

13

c

14

19104, United States

15

d

16

Pennsylvania, Philadelphia, PA 19104, United States

17

e

18

University of Pennsylvania, Philadelphia, PA, 19104, United States

Penn/CHOP Center of Excellence in Friedreich’s ataxia, Philadelphia, PA 19104, United States

Departments of Pediatrics and Neurology, Children's Hospital of Philadelphia, Philadelphia, PA

Departments of Pediatrics and Neurology Perelman School of Medicine, University of

Department of Medicine, Division of Hematology-Oncology, Perelman School of Medicine,

19 20

Keywords: Friedreich’s ataxia, genetic disease, parallel reaction monitoring, analytical

21

validation, biomarker sensitivity, biomarker specificity, nano-UHPLC, stable isotopes

22

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Friedreich’s ataxia (FA) is an autosomal recessive disease caused by an intronic

24

GAA triplet expansion in the FXN gene, leading to reduced expression of the mitochondrial

25

protein frataxin. FA is estimated to affect 1 in 50,000 with a mean age of death in the fourth

26

decade of life. There are no approved treatments for FA, although experimental approaches,

27

which involve up-regulation or replacement of frataxin protein, are being tested. Frataxin is

28

undetectable in serum or plasma, and whole blood cannot be used because it is present in long-

29

lived erythrocytes. Therefore, an assay was developed for analyzing frataxin in platelets, which

30

have a half-life of 10-days. The assay is based on stable isotope dilution immunopurification

31

two-dimensional

32

monitoring/mass spectrometry. The lower limit of quantification was 0.078 pg frataxin/µg protein

33

and the assay had 100% sensitivity and specificity for discriminating between controls and FA

34

cases. The mean levels of control and FA platelet frataxin were 9.4 ± 2.6 pg/µg protein and 2.4

35

± 0.6 pg/µg protein, respectively. The assay should make it possible to rigorously monitor the

36

effects of therapeutic interventions on frataxin expression in this devastating disease.

nano-ultrahigh

performance

liquid

chromatography/parallel

37

2


reaction

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38

There are > 40 rare genetic diseases that result from aberrant protein expression including,

39

Duchenne’s muscular dystrophy (DMD),1 spinocerebellar ataxia 1 (SCA-1),2 and Friedreich’s

40

ataxia (FA).3 Current approaches to developing therapies for these rare diseases primarily

41

involve increasing expression of the normal protein. The necessity for monitoring protein levels

42

was highlighted recently during the US Food and Drug Administration (FDA) fast-track approval

43

process for the drug eteplirsen (Exondys 51) to treat DMD.4 The lack of a rigorously validated

44

method to assess up-regulation of dystrophin levels in DMD patients made it difficult to show a

45

therapeutic response. This resulted in mobilization of advocate groups to help obtain FDA

46

approval, an approach that will be discouraged in the future.5

47

FA is an autosomal recessive disease estimated to affect 1 in 50,000.6 The abnormal

48

gene contains an expanded GAA repeat in intron 1, which causes transcriptional silencing and

49

reduced expression of the mitochondrial protein frataxin.7,8 Longer GAA repeats, which induce

50

a greater frataxin deficiency, are associated with earlier onset of the disease and increased

51

severity.9 A subset of patients (< 3%) have heterozygous GAA expansions coupled with a loss

52

of function mutation.3,10 The exact role of frataxin is not completely understood; it could serve

53

as an iron donor or regulate cysteine-derived persulfide formation during the assembly of

54

mitochondrial iron-sulfur complexes.11,12

55

activity of a number of mitochondrial enzymes involved in energy metabolism.13

Reduced frataxin expression results in decreased

56

FA is characterized by slowly progressive ataxia14 and hypertrophic cardiomyopathy.15

57

Symptoms generally appear during adolescence and patients on average become wheelchair-

58

bound 15.5-years after the disease onset.16,17 Lifespan is significantly reduced in FA with an

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average of death of 37-years, most commonly from cardiac-related pathologies.15 There are no

60

approved treatments for FA, although numerous experimental approaches are being tested,

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which primarily involve up-regulation of frataxin protein.17-21 We have developed a strategy to

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monitor improvements in mitochondrial metabolism using FA platelets.22,23 However, a more

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direct measure of frataxin expression is also required. This stimulated our development of a

3



Page 4 of 27

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sensitive and specific method for the quantification of mature platelet frataxin from FA patients.

65

This method also provides an approach to monitor new therapies that are being developed for

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rare genetic diseases of aberrant protein expression such as DMD24 and SCA-1.2

67 68 69 70 71 72 73

Figure 1. Sequence of full-length 23,135 Da frataxin (1-210) showing the mitochondrial

74

processing peptidase (MPP) cleavage sites to give mature frataxin (81-210, green).

75 76

The major FXN transcript encodes a full-length 210-amino acid form of frataxin (1-210;

77

Figure 1) with a molecular weight (MW) of 23,135 Da.

78

mitochondria where it is cleaved by mitochondrial processing peptidase (MPP) to the mature

79

active form of frataxin (MW = 14,268 Da, Figure 1).25-27 Mitochondrial mature frataxin (81-210)

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is not normally secreted into the circulation, which means that in vivo monitoring must be

81

conducted in blood cells rather than serum or plasma.28 Surprisingly, substantial amounts of

82

mature frataxin (81-210) are present in erythrocytes from controls (70 ng/mL blood)29 and FA

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cases (17 ng/mL blood)29 even though they lack mitochondria (Table 1). Erythrocytes have a

84

half-life of approximately 100-days (Table 1), which precludes the use of whole blood because

85

changes would not be detected during clinical trials. In contrast, platelets have 86% of the

86

mitochondria present in whole blood and a half-life of only 10-days (Table 1) making them an

87

attractive alternative.

88

4


It is rapidly translocated to the

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89

Table 1: Blood cell abundance,28 half-life,28 mitochondrial content,28 and reported mean frataxin

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levels.28,29 NR= not reported, DI = dipstick immunoassay, Mito = mitochondria.

Size (mm) Number (106/mL) Mito (per cell) Mito (106/mL) Mito (%) Half-life (days) Control levels (DI) FA case levels (DI)

Reticulo -cytes

Erythro -cytes

PlateLets

Granulo -cytes

PBMC

10

6-8

1-2

10-15

7-15

90

5400

280

4.65

3.1

2

0

6

2-30

12-30

173

0

1500

16

50

10

0

86

1

3

1

100

10

0.5

2-90

70 ng/mL blood 17 ng/mL blood

2.8 pg/µg protein 1.0 pg/µg protein

0.4 ng/mL bood

6.9 pg/µg protein 2.7 pg/µg protein

NR

NR

NR

91 92

A lateral flow immunoassay (dipstick immunoassay, DI) found a significant difference in

93

normal platelet frataxin (9.4 mAbs/µg protein) compared with FA platelet frataxin (3.2 mAbs/µg

94

protein), although there was considerable inter-individual overlap.28 Absolute values reported

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subsequently for FA peripheral blood mononuclear cells (PBMCs, monocytes and lymphocytes)

96

of 2.7 pg/µg protein28,29 (corresponding to 9.0 mAbs/µg protein28), made it possible to estimate

97

that low levels of frataxin are present in normal control platelets (2.8 pg/µg protein) and FA

98

platelets (1.0 pg/µg protein) (Table 1). The low overlapping levels,28 and the minimal amount of

99

available blood, led to our development of an immunopurification two-dimensional-nano-

100

ultrahigh performance liquid chromatography-parallel reaction monitoring/mass spectrometry (IP

101

2D-nano-UHPLC-PRM/MS) assay for platelet frataxin. We report the rigorous validation and

102

utility of the assay.

103

5



104 105

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EXPERIMENTAL SECTION Chemicals and materials.

Reagents and solvents were LC-MS grade quality unless

106

otherwise noted. [13C615N2]-lysine and [13C615N1]-leucine were from Cambridge Isotope

107

Laboratories (Andover, MA, USA). Anti-frataxin antibody (clone 1D9) was from LifeSpan

108

Biosciences, Inc. (Seattle, WA).

109

protease inhibitor cocktail, D,L-dithiothreitol (DTT), isopropyl-β-D-thiogalactoside (IPTG), bovine

110

serum albumin (BSA), human lysozyme, imidazole, triethanolamine, ethanolamine, and M9,

111

minimal salts, 5X powder, minimal microbial growth medium (M9 media) were purchased from

112

MilliporeSigma (Billerica, MA).

113

(Germantown, MD). LC grade water and acetonitrile were from Burdick and Jackson

114

(Muskegon, MI, USA).

115

Corporation (Grand Island, NY).

Dimethyl pimelimidate dihydrochloride (DMP), EDTA-free

Ni-NTA Agarose resin was purchased from Qiagen

Protein G magnetic beads were obtained from Life Technologies

116

Clinical samples. Blood samples were obtained from FA patients (Table S-1; average age

117

34.3) and unaffected control subjects (3 males, 4 females, average age 32.6). They all are

118

enrolled in an ongoing natural history study at the Children’s Hospital of Philadelphia (IRB # 01–

119

002609).

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Platelet isolation. Venous blood was drawn into 8.5 mL acid-citrate-dextrose Vacutainer

121

tubes. Blood (4 mL) was transferred to 15-mL polypropylene tubes and spun at 200 g for 13

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min at room temperature with no brakes as described previously.23 The platelet pellet was

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carefully washed twice with 0.5 mL platelet wash buffer (10 mM sodium citrate, 150 mM NaCl, 1

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mM EDTA, 1% (w/v) glucose, pH 7.4.) by spinning at 800 g for 5 min. All platelet samples were

125

immediately frozen at -80 ºC after preparation until analysis.

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Expression and purification of unlabeled and SILAC-labeled frataxin.

The coding

127

sequence of human frataxin (81-210) was amplified by PCR reaction from the FXN cDNA

128

plasmid (pTL1), which was a kind gift from Dr. M. Koenig.30 The amplified FXN fragment was

129

cloned into a pET21b plasmid and linked to the 6x histidine (His) sequence through the XhoI

6


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site at the 3’ end. The 6x His-tag fusion of frataxin was expressed in Escherichia coli BL21 DE3

131

in M9 media containing 1 mM MgSO4, 10 µM CaCl2, and 0.5% glucose with 100 mg/L ampicillin.

132

For expressing unlabeled frataxin, M9 medium was supplemented with 0.025% leucine and

133

lysine. For expressing stable isotope labeling by amino acids in cell culture (SILAC)-labeled

134

frataxin, M9 medium was supplemented with 0.025% [13C615N1]-leucine and [13C615N2]-lysine.

135

Cells were pre-cultured in 2 mL of corresponding M9 medium overnight at 37 °C (Figure S-1,

136

lanes 1 and 4). The next day, the bacteria were grown in 250 mL medium at 37°C to A 600nm =

137

0.9-1.0, and were induced for protein expression with 1 mM IPTG for 4 h at 30°C. Cells were

138

pelleted and resuspended in 10 mL of lysis buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10

139

mM imidazole, 10% glycerol, 2 mM β-mercaptoethanol, 2x protease inhibitor cocktail, 1mM

140

PMSF). Cell-pellets were then lysed by addition of 100 µL 10 mg/mL human lysozyme and

141

incubation on ice for 30 min followed by 6 x 20 sec probe sonication (Figure S-1, lanes 2, and

142

5). The samples were spun at 20,000 g for 30 min and the supernatants were incubated with

143

200 µL Ni-NTA resin for 1 h at 4˚C. The resin was washed twice with 10 mL of washing buffer

144

(50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole, 10% glycerol, 2 mM β-

145

mercaptoethanol, 2x protease inhibitor cocktail). Frataxin was eluted by incubating the resin

146

with 3 x 150 µL of elution buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 400 mM imidazole,

147

10% glycerol, 2 mM β-mercaptoethanol, 2x protease inhibitor cocktail).

148

unlabeled His-tagged frataxin (Figure S-1, lane 3). His-tagged SILAC-labeled frataxin (Figure S-

149

1, lane 6) was established as > 95% pure by SDS-PAGE and Coomassie Blue staining

The purity of the

150

To determine the unlabeled frataxin concentration, imidazole was removed by concentrating

151

100 µL of frataxin eluate in an Amicon 3 kDa 0.5 mL filter to < 15 µL and refilling the filter unit

152

with phosphate-buffered saline (PBS) at 4°C. This step was repeated 5 times and the purified

153

protein was suspended in 100 µL PBS. The frataxin concentration was determined by

154

measuring the absorption at 280 nm and using the extinction coefficient (λmax) = 26,930 M-1cm-1

7



155

(predicted by the amino acid sequence).31 The concentration of the unlabeled standard was

156

verified by comparing frataxin bands with bands from known amounts of BSA in a Coomassie

157

Blue stained SDS-PAGE gel. The concentration of the SILAC-labeled frataxin internal standard

158

was determined by comparing it with endogenous frataxin bands and BSA bands in a

159

Coomassie Blue stained SDS-PAGE gel. Frataxin standards were stored in PBS containing 1

160

mM DTT mixed with 50% glycerol at -20°C. For long term storage, the standards were kept at -

161

80°C.

162

DMP cross-linking of mAb with magnetic beads. Protein G magnetic beads were washed

163

twice with PBS before use. 80 µg of anti-frataxin mAb (1D9) was incubated with 10 mg of

164

protein G beads for 1 h at room temperature or 4˚C overnight. The antibody solution was

165

removed and beads were washed twice with 1 mL cross-linking buffer (0.2 M triethanolamine,

166

pH 8.2). Antibody bound protein G beads were incubated with 2 mL of freshly prepared DMP

167

solution (13 mg/mL in cross-linking buffer) at room temperature for 45 min on a rotator. DMP

168

solution was removed and beads were washed twice with 2 mL blocking buffer (0.1 M

169

ethanolamine, pH 8.2). Blocking buffer was removed and beads were washed twice with 2 mL

170

PBS. The beads were quickly rinsed with 2 mL 0.1 M glycine-HCl (pH 3) and washed three

171

times with 2 mL PBS. The beads were stored in 500 µL PBS at 4˚C. The antibody coupled

172

beads were shown to be effective 3 weeks after preparation with 0.02% sodium azide included

173

in PBS. For preparation of larger amounts, reagents were scaled up accordingly.

174

Cell lysate preparation. Frozen platelet pellets were lysed in 180 µL ice cold IP-lysis buffer

175

(50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM DTT) supplemented

176

with 1x complete protease cocktail and incubated on ice for 15 min. Cell lysates (4 µL) were

177

taken for protein concentration measurement by Bradford assay (Bio-Rad, Hercules, CA). A

178

SILAC-labeled frataxin internal standard (20 ng) was spiked in before addition of 20 µL of 10%

179

SDS solution to reach a 1% final SDS concentration. Samples were mixed and heated at 95˚C

180

for 5 min.

For viscous samples, pulse sonication was applied for 30 sec using a sonic

8


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181

dismembranator (Fisher). Samples were centrifuged at 16,000 g using a bench top centrifuge

182

for 15 min at room temperature. The supernatants were diluted 10 times by adding 1.8 mL of IP

183

lysis buffer with protease inhibitor before applying to Amicon Ultracel-50K filters (MilliporeSigma,

184

Burlington MA).

185

transferred to Amicon Ultracel-3K filter units and spun at 4000 g, 4 ˚C for 20 min until the

186

sample volume was concentrated to less than 300 µL. Samples were transferred to clean

187

Eppendorf Protein LoBind tubes (Eppendorf, Hamburg, Germany) for immunoprecipitation.

188

The filters were spun at 4000 g, 4˚C for 20 min.

Western blot and gel staining.

The flow-through was

Frataxin was detected by mAb anti-Frataxin (Abcam,

189

Cambridge, MA) and anti-mouse HRP (Santa Cruz Biotechnology, Dallas, TX). Western blots

190

were developed using ECL reagents. Gels were stained with colloidal Coomassie (Promega,

191

Madison, WI).

192

Intact protein analysis by high-resolution MS. Samples were infused into a Q Exactive

193

HF Hybrid Quadrupole-Orbitrap mass spectrometer equipped with a HESI probe with a flow rate

194

of 3 µL/min.

195

capillary temperature 300°C, ion polarity positive, S-lens RF level 60, in-source CID 80 eV,

196

resolution, 60,000, microscan,10.

MS operating conditions were as follows: spray voltage 4000 V, ion transfer

197

Immunoprecipitation and trypsin digestion. Samples were incubated with 0.3 mg of

198

DMP cross-linked anti-frataxin protein beads at 4˚C overnight in protein LoBind tubes. The

199

unbound samples were removed, the beads resuspended in 500 µL IP lysis buffer, and then

200

transferred to clean LoBind tubes. Samples were washed 3 times with 1 mL PBS and frataxin

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was eluted by incubating the beads with 100 µL elution buffer (300 mM acetic acid/10%

202

acetonitrile) with shaking for 15 min. The elutes were transferred to deactivated glass inserts

203

(Waters, Milford, MA) and dried under nitrogen flow.

204

aqueous NH4HCO3 (50 µL) containing 100 ng trypsin and incubated at 37˚C overnight before

205

LC-MS analysis.

9

Samples were dissolved in 25 mM



Surplus 5-day old human platelets were

206

Frataxin depletion of human platelet lysates.

207

provided by the Hospital of the University of Pennsylvania. Intact platelets (80 mg) were lysed

208

in 8 mL of IP lysis buffer containing 1 mM DTT and protease inhibitor. Samples were pulse-

209

sonicated for 1 min and the cell debris was removed by spinning at 16,000 g for 15 min. In the

210

first round of depletion, the supernatant was incubated with 40 µg anti-frataxin mAb 1D9 bound

211

to 6 mg protein G Dynabeads overnight followed by the removal of the beads, which removed

212

approximately 90% of endogenous frataxin. To maximize the depletion efficiency, lysates were

213

incubated a second time with 80 µg anti-frataxin mAb 1D9 bound to 12 mg protein G

214

Dynabeads for 20 h. The depletion efficiency was first examined by SDS-PAGE and western

215

blot by loading 70 µg of platelet lysates before and after depletion into a gel. LC-MS was then

216

employed to calculate the efficiency.

217

Method validation. Frataxin-depleted platelet protein and BSA were used for preparation of

218

calibration standards and quality controls (QCs).

219

spiking appropriate amounts of the frataxin standard to 500 µg frataxin-depleted platelet lysates

220

or 500 µg of BSA to make the final concentrations of 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5,

221

10, 20, 40 pg/µg protein. The preparation procedures for QC samples at concentrations of 0.25,

222

2.0 and 30 pg/µg protein were the same as for the calibration standards. Assay validation was

223

conducted according to US FDA guidance.32 Linearity of standard curves was evaluated from

224

0.078 pg/µg protein to 40 pg/µg protein. The lower limit of quantification (LLOQ) of 0.078 pg/µg

225

protein was defined as the lowest QC sample, which could be fitted to the calibration curve with

226

a residual of less than 10% when using BSA as matrix and peak area ratio of analyte to internal

227

standard deviating by < 15%. The accuracy and precision were determined on five replicates of

228

LLOQ, low quality control (LQC), middle quality control (MQC) and high quality control (HQC).

229

QC samples (n = 5) were analyzed on the same day (intraday) for the frataxin-depleted platelet

230

protein and BSA matrix. Interday assays were performed for each of the frataxin-depleted

10

Calibration standards were prepared by


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231

platelet protein QC samples on five different days (n=5) and on three different days (n=3) for the

232

BSA matrix QC samples.

233

2D-nano-UHPLC-PRM/HRMS. MS was conducted using a Q Exactive HF coupled to a

234

Dionex Ultimate 3000 RSLCnano with capillary flowmeter chromatographic systems (Thermo

235

Fisher Scientific, San Jose, CA, USA). The 2D system was setup as a pre-concentration mode

236

which was composed of a ten-port valve, one nanopump for delivering solvents to analytical

237

column, and a micropump for delivering solvents to trapping column. The 2D nanoLC system

238

was controlled by Xcalibur software from the Q-Exactive mass spectrometer. The LC trapping

239

column was an Acclaim PepMap C18 cartridge (0.3 mm × 5 mm, 100Å, Thermo Scientific) and

240

the analytical column was a C18 AQ nano-LC column with a 10 µm pulled tip (75 µm × 25 cm, 3

241

µm particle size; Columntip, New Haven, CT).

242

Samples (6 µL) were injected using the microliter-pickup injection mode. Loading solvent

243

was water/acetonitrile (99.7:0.3; v/v) containing 0.2% formic acid. In the sample loading step,

244

the valve stayed in loading position (1-2) with loading solvent at 10 µL/min for 3 min. In the

245

elution and analysis step, the valve stayed in injection position (1-10) at which the trapping

246

column was connected to the nanopump and the analytical column, and samples were back-

247

flushed into the analytical column. Washing of the trapping column using the nanopump was

248

continued until 5 min before the end of the run. Samples were eluted in the mass spectrometer

249

with a linear gradient at a flow rate of 0.4 µL/min. Solvent A was water/acetonitrile (99.5:0.5;

250

v/v) containing 0.1% formic acid, and solvent B was acetonitrile/water (98:2, v/v) containing

251

0.1% formic acid. The gradient on the analytical column was as follows: 2% B at the start, 5% B

252

at 9 min, 27% B at 27 min, 95% B at 31 min, held for 14 min, then re-equilibrated at 2% B from

253

48 min to 60 min.

254

Scientific). MS operating conditions were as follows: spray voltage 2800 V, ion transfer capillary

255

temperature 275°C, ion polarity positive, S-lens RF level 55, in-source CID 1.0 eV. Both full

256

scan and PRM were used. The full scan parameters were resolution 120,000, AGC target 3e6,

Nanospray was conducted using Nanospray Flex ion source (Thermo

11



257

maximum IT 100 ms, scan range 400-1300 m/z. The PRM parameters were resolution 60,000,

258

AGC target 2e5, maximum IT 80 ms, loop count 5, isolation window 2.0 Da, NCE 25. The PRM

259

were scheduled for 27.4 to 29.1 min for L136GGDLGTYVINK147, 25.5 to 27.7 min for

260

Q153IWLSSPSSGPK164, and 28.6 to 31.2 min for L198DLSSLAYSGK208.

261

Data analysis. Data analysis was performed using Skyline (MacCoss Laboratory, University of

262

Washington, Seattle, WA).33

263

unlabeled/light (L) peptide to SILAC-labeled/heavy (H) peptide was calculated by the Skyline

264

software and used for absolute quantification.

265

average L/H ratios of the three PRM transitions.

266

average of the three selected peptides.

267

transitions during method validation provided higher accuracy and precision compared with the

268

use of single PRM transitions. Although, the use of all three PRM transitions for quantification

269

could have increased the chance of interference, this was not observed during sample

270

validation or analysis of platelet samples. Statistical analysis was performed using GraphPad

271

Prism (v 5.01, GraphPad Software Inc., La Jolla, CA).

The peak area ratio of each PRM transition for each

The peptide ratios were calculated by the Frataxin levels were calculated from the

Interestingly, using the average of three PRM

272 273

RESULTS AND DISCUSSION

274

Selection of frataxin peptides and PRM transitions. The expressed and purified frataxin

275

standard was digested in-solution by trypsin, and the proteolytic peptides were analyzed in full

276

scan and PRM modes. Uniqueness of the tryptic peptides was verified by searching the BLAST

277

database in Skyline.33

278

precursor and all product ions assessed for signal intensity in the PRM/MS mode. The early

279

eluting R165YDWTGK171 and the late eluting N172WVYSHDGVSLHELLAAELTK192 tryptic

280

peptides exhibited signal fluctuations during LC-PRM/MS analysis. S81GTLGHPGSLEDTYER96

281

is the amino-terminal tryptic peptide derived from mature frataxin (Figure 1). A previous report

Six potentially useful peptides were selected (Figure S-2) and their

12


Page 12 of 27

Page 13 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


282

suggested that there could be a frataxin isoform with a five-amino acid addition (M76NLRK80) at

283

the amino terminus, which would also give rise to this tryptic peptide.34

284

81

285

for quantification were: L136GGDLGTYVINK147, Q153IWLSSPSSGPK164 and L198DLSSLAYSGK208

286

(Figure 2A).

Therefore,

GTLGHPGSLEDTYER96 was used only as a qualifying peptide and the three peptides used

287 (A)

288

y11+

y7+

y3+

LGGDLGTYVINK 136-147 MH22+ = 625.343 Da

289

NL: 3.77E6

y8+ y7+

y10+ y9+ y8+

y4+

QI W LSSPSS GPK LD L S S LAYSG K 153-164 MH22+ = 643.841 Da NL: 1.54E6

198-208 MH22+ = 577.309 Da NL: 1.42E6

290 291 (B)

y11+

y7+

y3+

LGGDLGTYVINK MH22+ = 636.367 Da

292

NL: 3.77E6

y10+ y9+ y8+

y8+ y7+

y4+

QI W LS SPS SGPK LDL S SLAYSGK MH22+ = 651.356 Da NL: 3.77E6

MH22+ = 591.841 Da NL: 3.77E6

293 294 295 296

Figure 2. Typical 2D-nano-UHPLC-PRM/MS chromatograms of three PRM transitions for three

297

peptides. (A) Endogenous peptides; (B) Stable isotope labeled peptides. The chromatograms

298

are from one sample used for standard curve construction. Light frataxin was 10 pg/µg and

299

heavy frataxin was 40 pg/µg.

300 301

Precursor ions of the peptides generated from pure protein standards presented 2-10 folder

302

high signals detected in full scan mode compared to signals of the most intense product ions

303

detected in PRM mode. However, the precursor ions had interfering signals or were even not

304

detected when the endogenous frataxin level was < 0.6 pg/µg protein in platelet protein. In

305

contrast, some product ions provided excellent signals for frataxin levels even below 0.05 pg/µg

306

protein.

307

endogenous frataxin (Figure 2A, Table S-2) and SILAC-labeled internal standard (Figure 2B,

Therefore, the three most intense product ions were used for PRM of both the

13



308

Page 14 of 27

Table S-2).

309

Preparation of frataxin protein standard and SILAC-labeled internal standard. Both the

310

endogenous and SILAC-labeled proteins were purified from E. coli with a two-amino acid linker

311

(LE) and His tag (HHHHHH) at the carboxyl terminus (Figure S-3). The linker to the protein and

312

the His-tag increased the mass of the unlabeled standard and SILAC-labeled standard by 1,055

313

Da and 1,269 Da, respectively (Figure S-3). This caused a slight reduction in mobility when

314

compared with the endogenous frataxin, which was extremely useful for evaluating the

315

efficiency of processing procedures by western blot (see below). Because commercial frataxin

316

protein standards are either the intermediate or the precursor forms of frataxin or have long

317

extended linker and tags, the purified E. coli expressed frataxin (> 95% purity) was used as an

318

authentic standard for assay validation. The protein concentration was determined by UV with

319

extinction coefficient = 26,930 M-1cm-1.31 The calculated frataxin concentration determined from

320

its UV absorbance was also consistent with the amount estimated by comparing it on SDS-

321

PAGE with the concentrations of BSA standards determined by Coomassie blue staining.

322

Trypsin digestion of SILAC-labeled frataxin revealed > 99.8% {heavy/(light + heavy)} labeling

323

efficiency based on the PRM transitions for the three peptides (Figure 2, Table S-2).

324

Interestingly, when the endogenous and SILAC-labeled frataxin proteins were mixed at a 1:1

325

ratio, the signal intensity for the precursor protonated molecule of the labeled peptide (such as

326

L198DLSSLAYSGK208) was generally lower than the corresponding endogenous peptide (Figure

327

S-4). This was because SILAC labeling of frataxin in E. coli resulted in significant M-1, M-2, M-3

328

isotopic peaks (Figure S-4). This was not due to the differences of isotopic composition in the

329

amino acid substrates and so most likely occurred through exchange of the [15N]-labeled leucine

330

by transamination during the labeling process. In support of this concept, transamination of

331

leucine is known to occur in E. coli.35

332

correlated with the number of leucine residues (1, 2, or 3; Figure S-4).

333

differences in isotopic composition of the labeled tryptic peptide standards did not cause any

Furthermore, the increase in M-1, M-2, and M-3

14


Fortunately, the

Page 15 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


334

interference in the PRM transitions that were used to monitor the endogenous tryptic peptides.

335

This meant that there was no effect on the assay sensitivity or specificity. However, incomplete

336

labeling of the SILAC frataxin standard meant that when standard curves were constructed for

337

each peptide separately, the slopes were slightly different, but the back calculated frataxin

338

values were highly consistent (see below). Different lots of the frataxin SILAC standard can be

339

prepared with no interference into the PRM channels used for analysis of frataxin. Therefore,

340

we do not anticipate any issues with assay performance so long as standard curves are

341

prepared for every new analytical run.

342

Sample processing and immunoprecipitation of frataxin. Frataxin forms high molecular

343

weight (> 600 kDa) oligomers in an iron-dependent manner.36-38 In order to effectively extract

344

frataxin from platelets before a high-speed centrifugation step that removes cell debris, 1% SDS

345

at 95 °C was used to denature cell lysates (Figure 3). Samples with 1% SDS were later diluted

346

ten times with IP-lysis buffer to accommodate antibody binding. Western blot analysis showed

347

that SDS-denaturation not only resulted in higher amounts of endogenous frataxin but more of

348

the SILAC-labeled frataxin added to the samples was also more soluble. Because our goal was

349

to quantify frataxin levels lower than 0.2 pg/µg, an enrichment step was necessary before LC-

350

MS detection (Figure 3). Numerous anti-frataxin monoclonal antibodies (mAbs) were tested

351

before it was found that mAb 1D9 had the strongest binding with the antigen. A substantial

352

number of additional proteins were captured by the mAb and were eluted with frataxin from the

353

beads (Figures S-5A, S-5B; lane 2). Most of these proteins (even those with molecular weights

354

< 50 kDa) were removed with a 50 kDa cut-off filter (Figures S-5A, S-5B; lane 3).

355

Light and heavy chains that arose from the mAb were not removed by filtration and they

356

caused suppression of ionization in source of the mass spectrometer limiting the assay

357

sensitivity. Covalently linking mAbs to the beads is known to prevents loss of heavy and light

358

mAb chains.39,40 DMP proved to be an efficient cross-linker so that it was possible to enrich

359

platelet frataxin by > 70 % by an overnight incubation with the DMP cross-linked magnetic

15



360

beads (Figures S-5A, S-5B; lane 4). The heavy and light mAb chains remained bound to the

361

beads but proteins that eluted from the non-covalently bound mAb were also detected even

362

after stringent washing (Figures S-5A, S-B; lane 4). Fortunately, they were readily removed by

363

the 50 kDa filter (Figures S-5A, S-5B; lane 5).

364 Platelet lysates SILAC frataxin

365 366 367

1% SDS - 95 °C denaturation and buffer dilution

368

50 kDa cut-off filter

370 371 372 373

fra ta xi n

369 S fra ILA ta C xi n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 27

Elution SILAC frataxin

frataxin

Trypsin digestion

374 375 376 377

DMP cross-linked frataxin mAb magnetic beads

2D-nano-UHPLCPRM/MS

Figure 3. Platelet frataxin sample processing procedure.

378 379

Method validation using IP-UHPLC-PRM/HRMS and SILAC-labeled frataxin.

Two-

380

rounds of frataxin depletion of platelets using an anti-frataxin antibody were performed.

381

Western blot and LC-MS analysis showed that more than >99.5% frataxin was depleted, while

382

the cell lysate protein composition remained unchanged. Calibration curves were constructed at

383

ten different concentrations spanning three orders of magnitudes (0.078-40.0 pg/µg protein)

384

using frataxin-depleted platelet lysates (500 µg) as the matrix. Because frataxin-depleted

385

platelet lysates are difficult to prepare a standard curve was also prepared by adding the

16


Page 17 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


386

standards to BSA (500 µg) as an alternative surrogate matrix. Overlapping linear standard

387

curves were obtained for each of the three peptides with r2 values > 0.999 for both the frataxin-

388

depleted platelet protein and the BSA matrix (Figures S-6A, S-6B, S-6C). Furthermore, similar

389

values were found when mean area ratios of analyte to internal standard for the three peptides

390

were plotted against frataxin concentrations (Figure S-6D).

391

The LLOQ was set at 0.078 pg/µg protein, which is 20-fold lower than the lowest amount ever

392

reported in FA platelets (Table 1). Accuracy and precision for the LLOQ using frataxin-depleted

393

platelet protein matrix were well within the limits of acceptance - intraday: (n=5) precision 5.1%,

394

accuracy 96.8%; interday (n=5), precision 3.7%, accuracy 101.0% (Table S-3A). Therefore, this

395

conservative LLOQ readily met the criteria required by the FDA of precision better than 15 %

396

and accuracy of between 85-115 %.

397 398 399

NL: 7.01E4

NL: 7.37E4

NL: 4.66E4

NL: 1.23E6

NL: 9.85E5

NL: 8.46E5

400 401 402 403 404 405 406

Figure 4. 2D-nano-UHPLC-PRM/MS chromatograms from an FA platelet sample with 2.1 pg/g

407

protein.

408

L136GGDLGTYVINK147, Q153IWLSSPSSGPK164, and L198DLSSLAYSGK208. (B) Three PRM/MS

409

transitions from the corresponding SILAC- labeled analogs (K = [13C615N2]-lysine, L = [13C615N1]-

410

leucine).

(A) Three PRM/MS transitions for endogenous platelet frataxin tryptic peptides –

17



Page 18 of 27

411

Additional

validation was performed using quality control (QC) samples at three different

412

concentrations according to FDA guidance41 with low (LQC, 0.25 pg/µg protein), middle (MQC,

413

2 pg/µg protein) and high (HQC, 30 pg/µg protein) QC samples prepared in frataxin-depleted

414

platelet protein. Precision for intraday (n=5) QC analysis was 2.0%-5.1% and accuracy was

415

90%-103.4%.

416

97.0%-101.1% (Table S-3A).

417

instead of frataxin-depleted platelet protein. Similar precision and accuracies were obtained:

418

intraday; (n=5) precision 1.0-1.5%, accuracy 94.5%-107.7%; interday (n=3), precision 1.3%-

419

3.1%, accuracy 97.5%-105.2% (Table S-3B). Our observation suggests that there is no matrix

420

effect for the range of frataxin concentration we examined. Therefore, calibration curves and

421

QCs prepared using BSA as surrogate matrix can be used for future analysis.

Precision of interday (n=5) QC analysis was 1.6%-4.8% and accuracy was Method validation was also conducted using BSA as matrix

422 423

Platelet frataxin levels in unaffected control subjects and FA cases.

To evaluate

424

clinical utility of the IP 2D-nanoUHPLC-PRM/MS method, frataxin levels were quantified in FA

425

platelets (n=7; Table S-1) and control platelets (n=7). Typical chromatograms for platelet

426

frataxin from an FA case are shown in Figures 4A and 4B, respectively. All samples were within

427

the linear range of the standard curves. The frataxin level (mean ± SD) in platelets from control

428

unaffected subjects was 9.4 ± 2.6 pg/µg protein. The mean level for FA cases was 74.5% lower

429

than controls at 2.4 ± 0.6 pg/µg (Figure 5A). There was no overlap between controls and FA

430

cases (Figure 5A), which meant the assay had 100% specificity and 100% sensitivity for

431

discriminating control and FA platelets. Despite the small sample size of FA cases (n = 7),

432

there was an inverse relationship of frataxin levels with the total number of GAA repeat lengths

433

(Figure 5B), which almost reached statistical significance (y=-0.0018x+5.13; r2 = 0.616,

434

p=0.067).

18


Page 19 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


435 (A)

436 437 438 439 440 441

(B)

442 443 444 445 446 447 448

Figure 5. Platelet frataxin levels. (A) Controls (mean ± SD) 9.4 ± 2.6 pg/µg protein. FA cases

449

2.4 ± 0.6 pg/µg protein. (B) Correlation of individual frataxin levels with sum of GAA repeat

450

lengths from both FXN alleles.

451 452

To evaluate the consistency for each peptide in the control and FA platelets, frataxin levels

453

determined from each of the individual tryptic peptides were compared with each other (Figures

454

S-7A, S-7B, S-7C). Excellent correlations were found for all three peptides from all the platelet

455

samples with r2 value ranging from 0.957-0.980.

456

SILAC-labeled frataxin standard corrected for the variations in IP and trypsin digestion

457

efficiency. It also confirmed that there were no post-translational modifications on the platelet

458

frataxin tryptic peptides that were analyzed.

This high consistency confirmed that the

459

19



460

Page 20 of 27

CONCLUSION

461

MS-based proteomics approaches have identified frataxin as one of the 4,497 human

462

platelet proteins.42-44 Similar approaches failed to detect frataxin as one of the 2,650 human

463

erythrocyte proteins,45 although it was detected by the DI assay (Table 1). This led to our

464

development of a more sensitive and specific stable isotope dilution IP 2D-nano-UHPLC-

465

PRM/MS method (Figure 3).

466

quantification of platelet frataxin because it corrected for losses during IP and trypsin

467

digestion.46,47

468

immunoprecipitation efficiency was noticed among different batches of samples, likely caused

469

by the filtration or antibody conjugation efficiency, or matrix effects. Although this variation had

470

very little impact on the LC-MS analysis, it reinforces the importance of using a SILAC internal

471

standard to compensate for these variations as well as the authentic protein for preparing

472

standard curves.

A SILAC-labeled standard (Figure S-3) facilitated accurate

The assay was highly precise and accurate (Table S-3).

Variation of

473

It is noteworthy that platelet frataxin levels in unaffected controls did not overlap with FA

474

cases (Figure 5A). Consequently, the assay had 100% sensitivity and 100% specificity for

475

discriminating the controls from FA cases. A previously reported DI method for platelet frataxin

476

had a sensitivity for distinguishing normal and FA platelet frataxin of 76.5% and a specificity of

477

only 66.7%. This was because the frataxin values for four of the nine controls overlapped with

478

seventeen of the FA cases and values from three of the FA cases overlapped with the nine

479

controls.28 The DI method also underestimated the amount of control platelet frataxin and FA

480

platelet frataxin (Table 1) by 70 % and 58 %, respectively.. Although, an inverse relationship

481

between FA platelet frataxin levels and total GAA repeat lengths was observed, it did not reach

482

statistical significance (Figure 5B, p=0.067). This likely reflects the small number of subjects

483

tested here and the relative inaccuracy of GAA repeat testing. However, the intercept on the y-

484

axis (GAA repeat lengths = 0) corresponded to 5.1 pg/µg protein (Figure 5B). Interestingly, 5.1

20


Page 21 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


485

pg/µg protein is close to the level found for the lowest unaffected normal control (6.1 pg/µg

486

protein) where total GAA repeat lengths were < 80. This suggests that it will be possible to

487

distinguish platelet frataxin levels in carriers with GAA repeats on only one FXN allele from both

488

FA cases and unaffected subjects.

489

The high sensitivity and specificity of the assay will also make it possible for the first time, to

490

rigorously evaluate the effect of new therapeutic strategies that are being employed to up-

491

regulate frataxin expression in human subjects.21 This will avoid controversial filings to the FDA

492

such as occurred recently with dystrophin (another platelet protein48) and the DMD drug

493

eteplirsen.4

494

relationship between levels in platelets and affected tissues.

495

modifications to the 2D nano-UHPLC-PRM/MS method because of the homology in amino acid

496

sequences of mouse (95.4% similarity) and monkey frataxin (98.5% similarity) when compared

497

with human frataxin.

Furthermore, mouse and monkey models can be employed to determine the This will require only minor

498 499

ASSOCIATED CONTENT

500

Supporting Information

501

The Supporting Information is available free of charge on the ACS Publications website

502 503

AUTHOR INFORMATION

504

Corresponding Author

505

*Tel: +1-215-573-9885. Fax: +1-215-573-9889. E-mail: [email protected]

506

Orcid

507

Ian A. Blair: 0000-0003-0366-8658

508

Author contributions

509

§

LG and QQW contributed equally to the work and should be regarded as co-first authors.

21



510

Notes

511

The authors declare no competing financial interests.

512

¶

513

Medicine, Washington University School of Medicine, St. Louis, MO, United States.

Current address: Agostinho Rocha, Center for Pharmacogenomics, Department of Internal

514 515

ACKNOWLEDGEMENTS

516

We gratefully acknowledge support of the Hamilton and Finneran families, the Penn

517

Medicine/CHOP Friedreich's Ataxia Center of Excellence, and NIH grants P42ES023720,

518

P30ES013508, R01FD006029, and UL1TR001878.

519 520

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