Liquid Chromatography-High Resolution Mass Spectrometry Analysis

Dec 22, 2017 - Penn/CHOP Center of Excellence in Friedreich's Ataxia, Philadelphia, Pennsylvania 19104, United States. § Departments of Pediatrics an...
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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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

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Disease Friedreich’s Ataxia

4 §

Qingqing Wang,a,b,

§

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Lili Guo,a,b,

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

6

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

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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,

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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

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

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

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

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

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

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

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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

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

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two-dimensional

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

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

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

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

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

nano-ultrahigh

performance

liquid

chromatography/parallel

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reaction

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Analytical Chemistry

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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mitochondrial iron-sulfur complexes.11,12

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

Reduced frataxin expression results in decreased

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

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

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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

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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

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

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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

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

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

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

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

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

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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

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

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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

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

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

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

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attractive alternative.

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It is rapidly translocated to the

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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

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

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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)

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

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

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

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

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

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

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utility of the assay.

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 EXPERIMENTAL SECTION Chemicals and materials.

Reagents and solvents were LC-MS grade quality unless

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

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

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Biosciences, Inc. (Seattle, WA).

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

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

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

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MilliporeSigma (Billerica, MA).

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

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(Muskegon, MI, USA).

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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

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

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

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

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002609).

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

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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

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immediately frozen at -80 ºC after preparation until analysis.

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Expression and purification of unlabeled and SILAC-labeled frataxin.

The coding

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The purity of the

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

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

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

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

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

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

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

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

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

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

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

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80°C.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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for 5 min.

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

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Analytical Chemistry

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

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

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

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Burlington MA).

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

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

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

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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,

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

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

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Madison, WI).

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

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

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of 3 µL/min.

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

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resolution, 60,000, microscan,10.

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

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

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

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

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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%

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

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

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

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LC-MS analysis.

9

Samples were dissolved in 25 mM

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Surplus 5-day old human platelets were

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Frataxin depletion of human platelet lysates.

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

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

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

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

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

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

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

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

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

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employed to calculate the efficiency.

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

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calibration standards and quality controls (QCs).

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

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

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

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

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

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

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

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

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

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

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

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

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Calibration standards were prepared by

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

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BSA matrix QC samples.

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

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Dionex Ultimate 3000 RSLCnano with capillary flowmeter chromatographic systems (Thermo

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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48 min to 60 min.

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

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

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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

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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

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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

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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

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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

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Analytical Chemistry

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

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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

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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 –

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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).

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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

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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

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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.

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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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