Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency

Clinical Mass Spectrometry xxx (xxxx) xxx

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Clinical Mass Spectrometry journal homepage: www.elsevier.com/locate/clinms

Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency Kazuhiro Aoki a, Adam D. Heaps b, Kevin A. Strauss b,c,⇑, Michael Tiemeyer a,d,⇑ a

Complex Carbohydrate Research Center, University of Georgia, Athens, GA, Greece Clinic for Special Children, Strasburg, PA, United States c Lancaster General Hospital, Lancaster, PA, United States d Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, Greece b

a r t i c l e

i n f o

Article history: Received 21 December 2018 Received in revised form 28 February 2019 Accepted 12 March 2019 Available online xxxx Keywords: Glycosphingolipid Ganglioside Plasma Mass spectrometry GM3

a b s t r a c t Background: Among Amish communities of North America, biallelic mutations of ST3GAL5 (c.694C > T) eliminate synthesis of GM3 and its derivative downstream a- and b-series gangliosides. Systemic ganglioside deficiency is associated with infantile onset psychomotor retardation, slow brain growth, intractable epilepsy, deafness, and cortical visual impairment. We developed a robust quantitative assay to simultaneously characterize glycan and ceramide moieties of plasma glycosphingolipids (GSLs) among ST3GAL5 c.694C > T homozygotes (n = 8), their heterozygous siblings (n = 24), and wild type control (n = 19) individuals. Methods: Following extraction and saponification of total plasma lipids, GSLs were purified on a tC18 cartridge column, permethylated, and subjected to nanospray ionization mass spectrometry utilizing neutral loss scanning and data-dependent acquisition. Plasma GSLs were quantified against appropriate synthetic standards. Results: Our method demonstrated linearity from 5 to 250 ll of plasma. Recovery of synthetic GSLs spiked into plasma was 99–104% with no matrix interference. Quantitative plasma GSL profiles discriminated among ST3GAL5 genotypes: GM3 and GD3 were undetectable in ST3GAL5 c.694C > T homozygotes, who had markedly elevated lactosylceramide (19.17 ± 4.20 nmol/ml) relative to heterozygous siblings (9.62 ± 2.46 nmol/ml) and wild type controls (6.55 ± 2.16 nmol/ml). Children with systemic ganglioside deficiency had a distinctive shift in ceramide composition toward higher mass species. Conclusions: Our quantitative glycolipidomics method discriminates among ST3GAL5 c.694C > T genotypes, can reveal subtle structural heterogeneity, and represents a useful new strategy to diagnose and monitor GSL disorders in humans. Ó 2019 Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL).

1. Introduction ST3GAL5 mediates the sialylation of lactosylceramide (LacCer) to produce GM3 (Neu5Ac a 2-3Galb1-4Glcb1-Cer), the root gan-

glioside for more complex downstream a- and b-series glycosphingolipids (GSLs, Fig. 1). Biallelic damaging variants of ST3GAL5 have been linked to systemic ganglioside deficiency in Old Order Amish, African-American, French, and Korean populations [1–5]. Complete

Abbreviations: GM3, monosialo-ganglioside GM3; Gal, galactose; Glc, glucose; GalNAc, N-acetylgalactosamine; Neu5Ac, sialic acid as N-5-acetylneuraminic acid; Cer, ceramide; GSL, glycosphingolipid; NSI, nanospray ionization; MS, mass spectrometry; Dp, degree of polymerization; Gb3, globotriaosylceramide (IUPAC-IUB: Gb3Cer); Gb3-D, deuterated Gb3; MSn, multidimensional mass spectrometry; CID, collision-induced dissociation; TIM, total ion mapping; NL, neutral loss; GlcCer, glucosylceramide; LacCer, lactosylceramide; Gb4, globotetraosylceramide (IUPAC-IUB: Gb4Cer); GM1b, monosialo-ganglioside GM1b (IUPAC-IUB: IV3-a-Neu5Ac-Gg4Cer); GD3, disialo-ganglioside GD3 (IUPAC-IUB: II3- a -(Neu5Ac)2-Gg2Cer); EGCase, endoglycosylceramidase; ESI-MS, electrospray ionization mass spectrometry; UPLC, ultra-high pressure liquid chromatography; ST3GAL5, CMP-Neu5Ac:Lactosylceramide alpha-2,3-sialyltransferase 5, previously known as SIAT9, SIATGM3S, ST3GalV, GM3-synthase. ⇑ Corresponding authors at: Clinic for Special Children, 535 Bunker Hill Rd., Strasburg, PA 17579, United States (K.A. Strauss). Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd., Athens, GA 30602, Greece (M. Tiemeyer). E-mail addresses: [email protected] (K. Aoki), [email protected] (A.D. Heaps), [email protected] (K.A. Strauss), [email protected] (M. Tiemeyer). https://doi.org/10.1016/j.clinms.2019.03.001 2376-9998/Ó 2019 Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL).

Please cite this article as: K. Aoki, A. D. Heaps, K. A. Strauss et al., Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency, Clinical Mass Spectrometry, https://doi.org/10.1016/j.clinms.2019.03.001

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K. Aoki et al. / Clinical Mass Spectrometry xxx (xxxx) xxx

unequivocally establishing the special dependence of neural tissues on ganglioside homeostasis [16–18]. Here, we report a novel method for quantification and structural analysis of plasma GSLs by nanospray ionization mass spectrometry (NSI-MS) and demonstrate that this method is broadly applicable to disorders of human GSL metabolism. Our optimized sample preparation is complemented by NSI-MS workflows that couple neutral loss scanning and data-dependent acquisition to allow simultaneous measurement of glycan structure and ceramide composition. We demonstrate the practical application of this approach by quantifying GSL changes in ST3GAL5 c.694C > T homozygotes compared to control populations, and suggest a broader role for the method in exploration of human gangliosidopathies. 2. Materials and methods 2.1. Patient samples The study was approved by the Penn Medicine-Lancaster General Hospital Institutional Review Board and study participants (or their parents) consented in writing. We collected EDTAanticoagulated plasma samples from ST3GAL5 c.694C > T homozygotes (n = 8), their heterozygous siblings (n = 24), and wild type control subjects (n = 19). The wild type control samples included plasma obtained from non-Amish (n = 11) and Amish (n = 8) individuals. Plasma was separated at 3260  g for 5 min and stored at 20 °C prior to analysis. 2.2. Materials

Fig. 1. Biosynthetic pathways for glycosphingolipid production. Lactosylceramide (LacCer) is produced by the glycosylation of ceramide (Cer) with glucose (Glc) and subsequent elongation with galactose (Gal). LacCer is a substrate for the production of multiple glycosphingolipid (GSL) types, including lacto- and neolacto-series, by extension with N-acetylglucosamine (GlcNAc), globo-series, by extension with Gal, or ganglio-series, by extension with N-acetylgalactosamine (GalNAc). Addition of sialic acid (Neu5Ac) to LacCer, catalyzed by ST3GAL5, generates GM3, the precursor for the production of all complex a- and b-series gangliosides. In the absence of ST3GAL5, the complex a- and b-series gangliosides are lost (red box) and LacCer is shunted toward production of externally sialylated o-series gangliosides as well as globo- and lacto-/neolacto-series GSLs.

We obtained GSL reference mixtures, high performance thinlayer chromatography silica gel 60 plates, and 50% sodium hydroxide (NaOH) from Matreya LLC (Pleasant Gap, PA, USA), EMD Millipore Chemicals (Darmstadt, Germany), and Fisher Scientific, respectively. Sep-Pak tC18 disposable extraction columns were obtained from Waters Corporation (Milford, MA, USA) and all other reagents were purchased from Sigma-Aldrich (St Louis, MO, USA). 2.3. Nomenclature Graphical representation of monosaccharide residues (as shown in Fig. 1) are consistent with the Symbol Nomenclature for Glycans (SNFG), which has been broadly adopted by the glycomics community [19]. 2.4. GSL extraction and preparation

a- and b-series GSL deficiency in humans manifests as neonatal hearing loss, stagnant brain growth, epileptic encephalopathy, cortical visual impairment, aberrant neural crest cell migration, and profound psychomotor retardation, underscoring the indispensible role of GSLs in CNS growth and development [6,7]. In contrast to the severe neurodevelopmental consequences of ST3GAL5 deficiency in humans, the St3Gal5 / knockout mouse has altered lymphocyte function and enhanced insulin sensitivity but relatively preserved CNS function; this divergence in phenotypes in part reflects the enzymatic redundancy of complex GSL production in mice as compared to humans [8,9]. Like humans, however, St3Gal5 / mice are deaf, which suggests a specific and indispensible role for GM3 in the mammalian cochlea [10,11]. Shortly after the discovery of ST3GAL5 deficiency in humans, mutations of the downstream enzyme, B4GALNT1 (GM2 synthase), were linked to a complex form of hereditary spastic paraplegia segregating among Old Order Amish, Kuwaiti, and Italian families [12–15]. Parallel studies of transgenic mice linked various neurological phenotypes to GSL synthetic defects,

GSLs were extracted from plasma by bringing the sample to a solvent mixture of chloroform (C), methanol (M), and water (W) equal to 4:8:3 (v/v/v), agitating for 30 min at room temperature in a closed glass tube, and removing protein by centrifugation [1,20]. We re-extracted the proteinaceous pellet three times with C/M/W (4:8:3), combined the resulting lipid extracts, and dried them under N2 stream. Analysis of GSLs harvested by these sequential extraction steps demonstrated that neutral and acidic GSLs were recovered from the proteinaceous pellets in the 1st and 2nd extractions, while the 3rd extraction yielded GSL at barely detectable levels. The dried lipid extract was saponified to remove glycerophospholipids by resuspending in 0.5 M potassium hydroxide in methanol/water (95:5, v/v) and incubating at 37 °C for 6 h. The reaction mixture was neutralized with 5% acetic acid on ice and adjusted with water to 50% aqueous methanol. The saponified material was subsequently loaded onto a Sep-Pak tC18 cartridge column (Waters, Sep-Pak Plus light, 145 mg resin,

Please cite this article as: K. Aoki, A. D. Heaps, K. A. Strauss et al., Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency, Clinical Mass Spectrometry, https://doi.org/10.1016/j.clinms.2019.03.001

K. Aoki et al. / Clinical Mass Spectrometry xxx (xxxx) xxx

#wat036805) using a glass syringe. The tC18 cartridge had previously been washed with methanol and pre-equilibrated with 1% acetic acid. Flow-through was collected during loading and reapplied to the column to increase recovery of GSLs. We then washed the column with 30 ml of 1% acetic acid, eluted GSLs with 3 ml of methanol, and dried them under N2 stream. Free fatty acids and sterols were removed from dried GSLs by extracting twice with cold hexane. 2.5. Permethylation Prior to MS analysis, we permethylated purified GSLs using methyliodide according to the method of Anumula and Taylor

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[21]. Known amounts of malto-series oligosaccharides (Dp3 and Dp4), permethylated with 13C-methyliodide, served as reference standards for GSL quantification [22]. 2.6. Quantification of plasma GSL, limit of detection, and matrix effects Prior to infusion into the mass spectrometer, we added 10 pmol each of 13C-labeled, permethylated Dp3 and Dp4 to permethylated plasma GSLs. We defined the limit of detection as the minimum amount of plasma GSL or synthetic standard that produced a signal-to-background ratio of three. Permethylated GSLs from plasma were diluted and re-analyzed by NSI-MS if the GSL peak intensity exceeded that of external calibrant by more than

Fig. 2. NSI-MS profiles of plasma GSLs. Representative full MS spectra are shown for analysis of GSLs prepared from plasma obtained from wild type control (+/+), ST3GAL5 c.694C > T heterozygous siblings, (M/+) and ST3GAL5 c.694C > T homozygous subjects (M/M). Each GSL glycan is associated with multiple peaks that reflect the heterogeneity of the ceramide portion of the molecule. Mass peaks attributed to GM3 are shaded in pink. Dp4, the maltotetraose calibrant peak is shaded red and was added in equal amount to all three samples.

Please cite this article as: K. Aoki, A. D. Heaps, K. A. Strauss et al., Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency, Clinical Mass Spectrometry, https://doi.org/10.1016/j.clinms.2019.03.001

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10-fold. To test for matrix effects on GSL quantification, we spiked plasma with synthetic globotriaosylceramide, Gb3-deuteride (Gb3-D, cat. # 1537, Matreya LLC, PA, USA). Gb3-D was also used as reference material to validate the use of Dp3 and Dp4 as external standards. Linearity of the method was validated by serial dilutions of control plasma GSLs across a 50-fold range. Gb3-D was also employed to assess the recovery of GSL by extraction from plasma; 20 ll of plasma was supplemented with 3, 25, or 75 pmol Gb3-D prior to extraction and the recovery of external Gb3-D standard was quantified relative to Gb3-D and relative to Dp3 and Dp4 standards. 2.7. Mass spectrometry of plasma GSLs Ionization efficiency is the key to detection sensitivity by NSIMS; we thus investigated the influence of three different solvent systems for infusion, each containing 1 mM NaOH: 50% methanol,

50% propanol, or methanol/2-propanol/1-propanol/water (16:3:3:2 by volume). For all three solvent systems, ions were detected as sodiated forms in positive ion mode. Permethylated GSLs were reconstituted in 50 ll of solvent, 10 pmol of 13C-permethylated maltooligosaccharide standard (Dp3 or Dp4) was spiked into the sample, and the mixture was analyzed by direct infusion into a linear ion trap mass spectrometer fitted with an Orbital Trap mass analyzer (Discovery LTQOrbitrap; Thermo Fisher Scientific, Waltham, MA) using a nanoelectrospray source at a syringe flow rate of 0.40–0.60 ll/min and capillary temperature of 210 °C [21,23–25]. The instrument was tuned with a mixture of permethylated GSLs and Dp standards in positive ion mode. For fragmentation by collision-induced dissociation (CID) in MS/MS and MSn, we used a normalized collision energy of 30–35%. Detection and absolute quantification of the prevalence of individual GSLs was accomplished using the total ion mapping (TIM)

Fig. 3. Quantification of plasma GSLs. GSLs were quantified in plasma obtained from wild type controls (+/+, n = 24), ST3GAL5 c.694C > T heterozygous siblings (M/+, n = 19), and ST3GAL5 c.694C > T homozygous subjects (M/M, n = 8). (A) Box-and-whisker plots present concentration ranges, means, and quartiles for GlcCer (1), LacCer (2), Gb3 (3), Gb4 (4), GM3 (5), GD3 (6), GM1b (7). (B) Direct comparative analysis of wild type, heterozygous, and homozygous subjects for LacCer, GM3, GD3, and GM1b levels. (C) Precursor/product ratios for the indicated lipids. ns, not significant; **P < 0.01, ****P < 0.0001.

Please cite this article as: K. Aoki, A. D. Heaps, K. A. Strauss et al., Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency, Clinical Mass Spectrometry, https://doi.org/10.1016/j.clinms.2019.03.001

K. Aoki et al. / Clinical Mass Spectrometry xxx (xxxx) xxx

and neutral loss scan (NL scan) functionality of the Xcalibur software package version 2.0 (Thermo Fisher Scientific), as previously described [23]. Briefly, for TIM, the m/z range from 600 to 2000 was automatically scanned in successive 2.8 mass unit windows with a window-to-window overlap of 0.8 mass units, which allow naturally occurring isotopes of each glycolipid species to be summed into a single response, thereby increasing detection sensitivity. Most GSLs were identified as singly, doubly, and triply charged, sodiated species [M+Na] in positive mode. Peaks were deconvoluted by charge state and summed for quantification. For NL scans, we defined an MS workflow in which the highest intensity peak detected by full MS was subjected to CID fragmentation. Major MS/MS fragment ions produced by CID of GSLs correspond to the neutral loss of the ceramide moiety, leaving an intact GSL oligosaccharide ion for subsequent fragmentation in MSn. Therefore, a NL scan workflow was designed to acquire MSn fragmentation if an MS/MS profile contained an ion with an m/z equivalent to loss of the most prevalent ceramide moiety. Following this data-dependent acquisition, the workflow returned to the full MS, excluded the parent ion just fragmented, and chose the peak of next highest intensity for the same MS/MS and MSn analysis. Thus GSL glycan profiles and MSn sequencing were rapidly acquired for complex mixtures of plasma GSL [1]. Glycomics data and metadata were obtained and are presented in accordance with MIRAGE standards and the Athens Guidelines [26,27].

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75 pmol, respectively, indicating complete recovery and lack of matrix effect (Supplemental Fig. 4). 3.4. Quantification of plasma GSL distinguishes ST3GAL5 c.694C > T homozygotes from heterozygotes and wild type controls Full MS spectra of plasma GSLs from wild type control (+/+), ST3GAL5 c.694C > T heterozygotes (M/+), and ST3GAL5 c.694C > T homozygotes (M/M) detect each GSL as a family of mass peaks, with each peak representing a different ceramide composition (Fig. 2). The wild type control population analyzed here was comprised of Amish (n = 8) and non-Amish (n = 11) individuals. Plasma lipid values for both populations were pooled together and considered as a single group after detecting no significant differences for any plasma lipid level between the two groups (Supplemental Tables 1 and 2). As expected, GM3 and GD3 were undetectable in plasma of ST3GAL5 c.694C > T approximately 3-fold higher than wild type control subjects (6.55 ± 2.16 nmol/ml). LacCer was also elevated in heterozygote plasma (9.62 ± 2.46 nmol/ml) compared to wild type control (45% increase, P < 0.01), providing a candidate biomarker of the carrier state (Fig. 3A, B). GM1b, the o-series, externally sialylated form of GM1, was approximately 3-fold higher in ST3GAL5 c.694C > T homozygotes (0.13 ± 0.03 nmol/ml versus 0.04 ± 0.02 nmol/ml), who also had slightly elevated plasma levels

3. Results 3.1. Optimum direct infusion conditions for GSL detection by mass spectrometry GSLs diluted in the 1 mM NaOH in methanol/2-propanol/1-pro panol/water (16:3:3:2 by volume) solvent produced more than 5-fold stronger signals than when diluted in standard solvent (50% methanol) or in its slightly more non-polar version (50% propanol), reflecting the relative hydrophobicity of ceramide (Supplemental Fig. 1). The 16:3:3:2 system was thus used for all subsequent analyses. 3.2. Extraction and detection of plasma GSLs exhibits a linear response and low detection limit Major plasma GSL concentrations produced linear responses over the entire range of plasma volumes assayed (Supplemental Figs. 2 and 3), with individual correlation coefficients 0.993. Synthetic GSL standards diluted 1:5, 1:10, and 1:50 produced quantitatively similar results (data not shown). Based on 3-fold signal-tobackground threshold, the quantitation limit of our full MS method was 0.02 pmol, allowing accurate measurement of major and minor plasma GSL species across all sample volumes and concentrations tested. However, the background detected in MSn fragmentation spectra is essentially zero, and thus the limit of detection was neither reached in practice nor determinable in principle. 3.3. Recovery of exogenous GSL demonstrates lack of matrix interference To assess recovery of GSLs and the potential for biomatrix interference, 20 ll of plasma was supplemented with 3, 25, or 75 pmol Gb3-D prior to extraction and purification (n = 3 replicates per condition). Following work-up and permethylation, mixtures of plasma GSL, external calibrant, and Gb3-D were analyzed by NSIMS. The recovery of synthetic Gb3-D (based on reported purity of >98%) was 103.8 ± 2.3%, 102.7 ± 1.2%, 99.2 ± 2.5% for 3, 25, and

Fig. 4. Plasma LacCer ceramide heterogeneity. (A) Representative zoom spectra for the mass range containing LacCer ceramide forms are presented for a wild type control (+/+) and an ST3GAL5 c.694C > T homozygous subject (M/M). The m/z values at which LacCer species were detected are shown along the x-axis. (B) Summary of the mean relative abundances of LacCer ceramide forms in wild type controls and ST3GAL5 c.694C > T homozygous subjects. Pie slices are color-coded to match the m/ z values in panel A.

Please cite this article as: K. Aoki, A. D. Heaps, K. A. Strauss et al., Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency, Clinical Mass Spectrometry, https://doi.org/10.1016/j.clinms.2019.03.001

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of Gb3 and Gb4, reflecting diversion of LacCer to alternative synthetic pathways (Table 1 and Fig. 3A, B). The absolute levels of GM3, expressed as nmol/ml plasma were not able to distinguish wild type controls from ST3GAL5 c.694C > T heterozygotes. Nonetheless, elevated levels of LacCer measured in heterozygotes suggest impaired flux through the biosynthetic pathway initiated by ST3GAL5. Therefore, we assessed precursor/ product relationships for the immediate product of ST3GAL5 (GM3) and for the next step in the pathway (GD3) by calculating LacCer/GM3 and LacCer/GD3 ratios for each control and heterozygous individual (Table 1 and Fig. 3C). Both of these precursor/product parameters achieved greater significance for distinguishing heterozygotes from control (P < 0.0001) than did the absolute level of LacCer (P < 0.01). However, calculation of LacCer/GM1 ratios demonstrated no significant differences between the three genotypes (Table 1 and Fig. 3C), indicating that increased LacCer in heterozygotes and homozygotes is shuttled through the biosynthetic pathway for GM1b with equal efficiency; i.e.: increased LacCer generates a proportional increase in GM1b regardless of ST3GAL5 status. 3.5. ST3GAL5 c.694C > T homozygotes have distinctive ceramide profiles Full MS spectra capture both the abundance of specific GSL glycan headgroups as well as the ceramide heterogeneity associated with each glycan. Ceramide profiles in ST3GAL5 c.694C > T homozygotes were markedly shifted toward higher mass species (Fig. 2); this shift was especially noticeable for LacCer (ANOVA P < 0.0001, Fig. 4 and Table 2). In all subjects, LacCer ceramide profiles were

dominated by two major species, d18:1;C16:0 detected at m/ z = 1010.8 and d18:1;C24:1 detected at m/z = 1120.9. In plasma of ST3GAL5 c.694C > T homozygotes, the d18:1;C24:1 form (m/ z = 1120.9) was 29.9 ± 6.9% of the abundance of the d18:1;C16:0 form (m/z = 1010.8) as compared to 12.6 ± 3.6% in wild type control and heterozygotes. The relative abundance of other less prevalent ceramide forms (d18:1;C18:0 at m/z = 1038.8, d18:1;C20:0 at m/ z = 1066.8, d18:1;C24:0 at m/z = 1122.9) were also increased, by 47.5–140.8% in ST3GAL5 c.694C > T homozygotes compared to control. While each of these increases were statistically significant (P > 0.01 to >0.0001), the slight increase (12.2%) in the d18:1; C22:1 form was not (Table 2). In order to further characterize the shift in the profile of the ceramide moieties of ST3GAL5 c.694C > T homozygotes, we carried out targeted MSn analyses to more accurately map ceramide compositions. We identified a characteristic ion at m/z = 278.29 resulting from fragmentation of permethylated ceramides which corresponds to d18:1 sphingosine produced from d18:1;C16:0 LacCer at m/z = 1010.8. We also performed MSn analysis of permethylated LacCer detected at m/z = 1152.9, which possesses a ceramide of additional mass 142.1 Da compared to the LacCer at m/z = 1010.8, indicating that the ceramide of the LacCer at m/z = 1152.9 is a d18:0;C26:0 or a d18:1;hC24:0 hydroxylated form. Fragmentation of this LacCer at m/z = 1152.9 produced the characteristic ion at m/ z = 278.29 (derived from d18:1 sphingosine), confirming that the LacCer at m/z = 1152.9 carries 2-hydroxylated C24 fatty acid. TLC analysis of plasma GSLs was consistent with this assignment since we observed two distinct LacCer bands corresponding to nonhydroxylated and hydroxylated fatty acid compositions. Furthermore, we used a-GalCer (t18:1;C26:0, Avanti Polar Lipid) to define

Table 1 Plasma glycosphingolipid levels in control (+/+), ST3GAL5 c.694C > T heterozygous (M/+), and ST3GAL5 c.694C > T homozygous (M/M) subjects.

a b c d e

Glycosphingolipid (nmol GSL per ml plasma)

+/+ (n = 19) Mean ± SD

M/+ (n = 24) Mean ± SD

M/M (n = 8) Mean ± SD

ANOVA P-value

Tukey Multiple Comparison Testa +/+ vs. M/+

+/+ vs. M/M

M/+ vs. M/M

CMH (GlcCer) CDH (LacCer) CTH (Gb3Cer) CQH (Gb4Cer) GM3 GD3 GM1 LacCer/GM3 ratio LacCer/GD3 ratio LacCer/GM1 ratio

5.39 ± 1.65 6.55 ± 2.16 2.18 ± 0.97 1.27 ± 0.55 4.61 ± 1.82 0.14 ± 0.06 0.04 ± 0.02 1.5 ± 0.3 47.2 ± 9.6 162.6 ± 37.6

7.20 ± 1.93 9.62 ± 2.46 3.09 ± 0.90 1.88 ± 0.44 4.84 ± 1.34 0.13 ± 0.04 0.06 ± 0.02 2.0 ± 0.5 80.7 ± 26.4 160.8 ± 48.4

3.78 ± 1.44 19.17 ± 4.20 4.92 ± 1.33 3.58 ± 0.98 ndb Nd 0.13 ± 0.03 ncc ncc 162.6 ± 37.6

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 nad nad ns

**

ns

****

**

****

****

*

****

***

**

****

****

ns ns

****

****

****

****

**

****

****

****e

ncc ncc ns

ncc ncc ns

*

**

***

****

ns

****

ns, not significant; P < 0.05; <0.01; <0.001; <0.0001. nd, not detected. nc, not calculable, division by zero. na, not applicable. Significance levels for GSL ratios were calculated using the log10 of ratio values.

Table 2 Ceramide composition of plasma LacCer (CDH) in control (+/+), ST3GAL5 c.694C > T heterozygous (M/+), and ST3GAL5 c.694C > T homozygous (M/M) subjects.

a b

Ceramide Composition (Percent of Total Profile)

+/+ (n = 19) Mean ± SD

M/+ (n = 24) Mean ± SD

M/M (n = 8) Mean ± SD

ANOVA P-value

Tukey Multiple Comparison Testa +/+ vs. M/+

+/+ vs. M/M

M/+ vs. M/M

d18:1,c16:0b d18:1,c18:0 d18:1,c20:0 d18:1,c22:1 d18:1,c22:0 d18:1,c24:1 d18:1,c24:0

64.0 ± 6.0 2.3 ± 0.6 2.1 ± 0.6 2.8 ± 0.7 8.0 ± 2.6 12.9 ± 1.9 7.9 ± 1.6

62.6 ± 5.0 2.7 ± 0.6 2.2 ± 0.7 3.0 ± 1.3 8.2 ± 2.6 12.9 ± 1.3 8.4 ± 1.6

42.5 ± 3.3 5.5 ± 1.6 5.1 ± 1.2 3.2 ± 0.6 11.7 ± 1.2 19.4 ± 2.8 12.5 ± 1.9

<0.0001 <0.0001 <0.0001 ns 0.0014 <0.0001 <0.0001

ns ns ns ns ns ns ns

****

****

****

****

****

****

ns

ns

**

**

****

****

****

****

ns, not significant; *P < 0.05; ** <0.01; ***<0.001; **** <0.0001. lipid nomenclature indicates chain length and desaturation of the sphingosine (d) and of the fatty acid (c) in amide linkage to the sphingosine amine.

Please cite this article as: K. Aoki, A. D. Heaps, K. A. Strauss et al., Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency, Clinical Mass Spectrometry, https://doi.org/10.1016/j.clinms.2019.03.001

K. Aoki et al. / Clinical Mass Spectrometry xxx (xxxx) xxx

a characteristic fragment ion for detecting t18:0 phytosphingosine at m/z = 310.3 and were unable to detect its presence in plasma GSLs. The increased abundance of longer chain N-acyl ceramides observed for LacCer of ST3GAL5 c.694C > T homozygotes led us to survey the distribution of ceramides linked to other GSL glycan headgroups. We extracted peak intensities for the dominant short and long chain form(s) of the seven most abundant GSLs. Peak intensities associated with ceramides bearing N-acyl chains with 20 or fewer carbons were summed together as short forms while the intensities associated with ceramides bearing N-acyl chains with 22–24 carbons were summed together as long forms (Fig. 5 and Table 3). The ceramides of GlcCer and LacCer were significantly enriched in long acyl chains when comparing ST3GAL5 c.694C > T homozygotes to wild type controls or heterozygotes (P < 0.0001), while the ceramide distributions of the globo-series GSLs were conserved across sample types. GM1 ceramides were also enriched in long N-acyl chains in heterozygotes compared to wild type controls (P < 0.05), but enrichment in homozygotes did not reach statistical significance. The ceramides of the disialo-ganglioside GD3 did not distinguish wild type from heterozygotes and the ceramides of GM3 were observed to be enriched in short N-acyl chains compared to wild type. Thus, the only ceramide profile that robustly distinguished wild type control from heterozygotes was the relative decrease in N-acyl chain length for GM3 (P < 0.0001).

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structural information about ceramide heterogeneity. By coupling TIM and NL scanning with data-dependent acquisition of MSn fragmentation profiles, our workflow achieved accurate quantitation and reliable structural assignment. Based on the method’s linearity, detection limit, and lack of matrix effects, it serves as a robust tool for investigating GSLs in normal and pathological samples.

4. Discussion 4.1. Validation of intact GSL analysis and quantitative mass spectrometric approach Many current methods for quantifying GSL glycans in biological samples achieve high sensitivity by reductively tagging glycans with fluorophores following their enzymatic release from ceramide moieties. Endoglycosylceramidase (EGCase, also known as ceramide glycanase) is widely used to release GSL glycans from their ceramide moieties prior to structural analysis [28,29]. Ceramides are structurally heterogeneous, composed of one of several sphingosine bases in amide linkage to a variety of fatty acids. EGCase simplifies the complexity of the GSL glycan analyte by removing confounding ceramide heterogeneity, but the approach has limitations. Efficiency of EGCase-mediated glycan release decreases with increasing sialylation or sulfation of the neutral glycan core [28,29] and, although released glycans can be directly analyzed by several techniques (e.g. MALDI-TOF/MS, ESI-MS, HPLC with fluorophore derivatization), such methods typically do not allow for simultaneous quantitation of neutral and acidic species [25,30–35]. Our analytical method circumvents these limitations, allowing neutral and acidic glycans to be characterized simultaneously while preserving ceramide structural integrity, which may have biological relevance for certain disease states. To simultaneously prepare neutral and acidic GSLs for analysis, we used the common practice of permethylation prior to mass spectrometry [21,23]. Permethylation caps the carboxylic acid moiety of acidic GSLs (usually a sialic acid) with a methyl group, resulting in charge neutralization; permethylated acidic GSLs ionize and fragment with similar efficiency to neutral GSLs, allowing simultaneous quantitation of acidic and neutral GSLs in a single analytical run [22]. Moreover, modern MS systems have mass resolution sufficient to clarify the specific sphingosine and fatty acyl composition of each glycan-associated ceramide, information that is otherwise lost when glycan profiles are analyzed after EGCase digestion. We took advantage of the NSI-MS platform, which achieves high sensitivity for detection of permethylated glycoconjugates, simultaneously measures neutral and acidic GSLs, and reveals

Fig. 5. Heterogeneity of plasma GSL ceramides. For each of the indicated GSLs, MS peak intensities associated with the most abundant ceramides bearing short (c16c20, black pie slices) or long (c22-24, grey pie slices) N-acyl chains were summed together. The summed intensities for short or long N-acyl chains were normalized to the total intensity and are plotted as pie charts to compare the relative abundance of ceramide forms across wild type control (+/+), ST3GAL5 c.694C > T heterozygotes (M/+), and homozygotes (M/M). ns, not significant; ****P < 0.0001; nd, not detected; *P < 0.05.

Please cite this article as: K. Aoki, A. D. Heaps, K. A. Strauss et al., Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency, Clinical Mass Spectrometry, https://doi.org/10.1016/j.clinms.2019.03.001

8

K. Aoki et al. / Clinical Mass Spectrometry xxx (xxxx) xxx

Table 3 Ceramide acyl chain composition of plasma glycosphingolipids in control (+/+), ST3GAL5 c.694C > T heterozygous (M/+), and ST3GAL5 c.694C > T homozygous (M/M) subjects. Ceramide Composition (% Distribution between Short and Long Acyl Chains)

a b c d

+/+ (n = 19) Mean ± SD

M/+ (n = 24) Mean ± SD

M/M (n = 8) Mean ± SD

ANOVA P-value

Tukey Multiple Comparison Testa +/+ vs. M/+

+/+ vs. M/M

M/+ vs. M/M

CMH (GlcCer)

Shortb; d18:1, c16:0-c20:0 Long; d18:1, c22:0-c24:0

16.1 ± 1.7 83.9 ± 1.7

17.1 ± 1.8 82.8 ± 1.8

31.7 ± 7.6 68.3 ± 7.6

<0.0001

ns

****

****

CDH (LacCer)

Short; d18:1, c16:0-c20 Long; d18:1, c22:0-c24:0 and c24:1

68.4 ± 5.4 31.6 ± 5.4

67.5 ± 4.5 32.5 ± 4.5

53.2 ± 4.6 46.8 ± 4.6

<0.0001

ns

****

****

CTH (Gb3Cer)

Short; d18:1, c16:0-c20:0 Long; d18:1, c22:0-c24:0

52.2 ± 3.5 47.8 ± 3.5

52.8 ± 3.8 47.2 ± 3.8

52.4 ± 11.3 47.6 ± 11.3

ns

ns

ns

ns

CQH (Gb4Cer)

Short; d18:1, c16:0-c20:0 Long; d18:1, c22:0-c24:0

51.5 ± 4.6 48.5 ± 4.6

46.2 ± 14.6 53.8 ± 14.6

54.5 ± 5.2 45.4 ± 5.2

ns

ns

ns

ns

GM3

Short; d18:1, c16:0-c20:0 Long; d18:1, c22:0-c24:0

36.9 ± 5.0 63.1 ± 5.0

43.2 ± 3.5 56.8 ± 3.5

ndc nd

nad

****

na

na

GD3

Short; d18:1, c16:0-c20:0 Long; d18:1, c22:1 and c22:0-c24:0

52.8 ± 4.6 47.2 ± 4.6

49.4 ± 16.6 50.6 ± 16.6

nd nd

na

ns

na

na

GM1

Short; d18:1, c16:0-c20:0 Long; d18:1, c:22:0-c:24:0 and c24:1

92.6 ± 11.6 7.40 ± 11.6

75.3 ± 26.7 24.7 ± 26.7

82.2 ± 10.5 17.8 ± 10.5

<0.05

*

ns

ns

ns, not significant; *P < 0.05; ** <0.01; ***<0.001; **** <0.0001. lipid nomenclature indicates chain length and desaturation of the sphingosine (d) and of the fatty acid (c) in amide linkage to the sphingosine amine. nd, not detected. na, not applicable.

4.2. GM3 is undetectable in ST3GAL5 c.694C > T homozygotes Quantitative changes of permethylated GSLs we detected by NSI-MS are in good agreement with previous studies of ST3GAL5 c.694C > T homozygotes that utilized HPLC of fluorescently tagged glycans released by EGCase treatment [4]. However, NSI-MS allowed certain aspects of the GSL profile to be resolved in greater detail. Unlike previous reports, we detected changes in neutral and acidic GSLs in ST3GAL5 c.694C > T heterozygotes, who had elevated levels of LacCer relative to wild type individuals. Accordingly, GM3 and GD3 constitute a lower proportion of the circulating GSL pool, which, in these subjects, serves as a distinctive biomarker of their heterozygous state. Recently, the detection of GM3 in plasma of ST3GAL5 c.694C > T homozygotes was reported at levels approximately 1% of wild type controls using a method that employed targeted derivatization of sialic acids followed by UPLC-MS/MS analysis [32]. While LCbased analytic approaches can offer increased sensitivity due to analyte concentration, our results, and those of Simpson, et al., are not consistent with this previous report, despite the fact that the dynamic range of our method is sufficient to detect GM3 at 1/100th of wild type control levels [4]. For example, we detect plasma GM1 levels at approximately 1% of GM3 levels in wild type controls, also consistent with Simpson et al. [4]. Retrospective inspection of the MS/MS fragmentation obtained for ST3GAL5 c.694C > T homozygote plasma samples, which is at least an order-of-magnitude more sensitive than analysis at the full MS levels, reveals the presence of fragments consistent with the presence of GM3 at detectable but unquantifiable levels. At this extremely low level of abundance, well below that reported by the UPLC-MS/MS method, it is not possible to rule out that the detected GSL is LacCer sialylated by another sialyltransferase in the absence of ST3GAL5, perhaps even an a 2–6 siayltransferase acting with extremely low efficiency on a poor but highly abundant substrate (LacCer). The discrepancy between the levels of GM3 reported by the UPLC-MS/MS method and by our NSI-MS method remains to be resolved. We note, however, that quantification of GM3 in the UPLC-MS/MS method was achieved by spiking plasma samples with known amounts of deuterated, synthetic GM3 as an external calibrant. If the isotopic distribution of the labeled GM3 was 99% fully deuterated and 1% 1H, the GM3 detected in ST3GAL5

c.694C > T homozygotes might have come from the calibrant. Since the method we report here takes advantage of the uniform molar responses achieved by permethylation, it does not require the acquisition of synthetic gangliosides labeled with stable isotopes as standards and is, therefore, not subject to the confounding consequences of synthetic or isotopic contaminants [22].

4.3. Ceramide profiles shift in response to altered GSL diversity Using NSI-MS analysis, we unexpectedly discovered that ST3GAL5 c.694C > T homozygotes express ceramide moieties with longer chain length and alternative saturation. Such structural changes are expected to alter membrane fluidity and lipidprotein interactions within the bilayer of their cell of origin [36– 39]. A similar shift in ceramide structure, most apparent for LacCer, was recently found in fibroblasts from non-Amish subjects with ST3GAL5 deficiency (i.e. ‘‘Salt and Pepper Syndrome”) and zebrafish st3gal5 morphant embryos [1]. Nevertheless, very little is known regarding the impact of ceramide heterogeneity on the fate and function of GSLs released into plasma. In the absence of ST3GAL5 activity, our data demonstrate that LacCer is shunted into alternative biosynthetic pathways (e.g. globo-series GSLs). However, the marked accumulation of LacCer in plasma reveals limited synthetic capacity of alternative pathways relative to the normal biosynthetic flux toward production of GM3. Additionally, the skewing of ceramide profiles toward longer N-acyl chains in GlcCer and LacCer, but not in other GSLs, indicates an unappreciated influence of lipid structure on GSL biosynthesis. Other factors might contribute to the high steadystate LacCer levels observed in ST3GAL5 c.694C > T homozygotes. Endocytic and lysosomal degradation pathways that contribute to GSL homeostasis might have evolved to more effectively reutilize complex GSLs rather than small neutral GSLs such as LacCer. If so, ceramides of unprocessed LacCer could become populated by otherwise minor components that accumulate over time. Observed shifts in ceramide structure could also represent a compensatory mechanism (e.g. altered ceramide synthase expression patterns) to modulate membrane biophysics in the absence of GM3. Each of these possibilities can be examined in the future using cell-based models and the analytical method described herein.

Please cite this article as: K. Aoki, A. D. Heaps, K. A. Strauss et al., Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency, Clinical Mass Spectrometry, https://doi.org/10.1016/j.clinms.2019.03.001

K. Aoki et al. / Clinical Mass Spectrometry xxx (xxxx) xxx

In summary, the constellation of undetectable GM3 and GD3, elevated LacCer and GM1b, and altered ceramide composition represents a biochemical fingerprint of recessive ST3GAL5 (GM3 synthase) deficiency. We developed, optimized, and validated an NSIMS method that reliably reveals this pattern in human samples and is thus useful for the diagnosis and monitoring of systemic GSL deficiency. Our method is sufficiently robust and granular to apply to a wide variety of human disease states linked to aberrant GSL metabolism and should have broad utility in this sphere of application. Acknowledgments This work was supported by grants from the National Institutes of Health, USA (NIGMS P41GM103490 to M.T. and Common Fund R21AI129873 to K.A.) and from the W.M. Keck Foundation (to M. T., K.A., K.S.). The authors thank Dr. D. Holmes Morton for his collaboration and valuable clinical insights during the early phases of sample acquisition and study design. Conflict of interest None of the authors has any conflicts of interest to disclose. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.clinms.2019.03.001. References [1] L. Boccuto, K. Aoki, H. Flanagan-Steet, C.F. Chen, X. Fan, F. Bartel, et al., A mutation in a ganglioside biosynthetic enzyme, st3gal5, results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation, Hum. Mol. Genet. 23 (2014) 418–433. [2] K. Fragaki, S. Ait-El-Mkadem, A. Chaussenot, C. Gire, R. Mengual, L. Bonesso, et al., Refractory epilepsy and mitochondrial dysfunction due to gm3 synthase deficiency, Eur. J. Hum. Genet. 21 (2013) 528–534. [3] J.S. Lee, Y. Yoo, B.C. Lim, K.J. Kim, J. Song, M. Choi, J.H. Chae, Gm3 synthase deficiency due to st3gal5 variants in two korean female siblings: Masquerading as rett syndrome-like phenotype, Am. J. Med. Genet. A 170 (2016) 2200–2205. [4] M.A. Simpson, H. Cross, C. Proukakis, D.A. Priestman, D.C. Neville, G. Reinkensmeier, et al., Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of gm3 synthase, Nat. Genet. 36 (2004) 1225–1229. [5] H. Wang, A. Bright, B. Xin, J.R. Bockoven, A.S. Paller, Cutaneous dyspigmentation in patients with ganglioside gm3 synthase deficiency, Am. J. Med. Genet. A 161A (2013) 875–879. [6] T.A. Li, R.L. Schnaar, Congenital disorders of ganglioside biosynthesis, Prog. Mol. Biol. Transl. Sci. 156 (2018) 63–82. [7] H. Wang, A. Wang, D. Wang, A. Bright, V. Sency, A. Zhou, B. Xin, Early growth and development impairments in patients with ganglioside gm3 synthase deficiency, Clin. Genet. 89 (2016) 625–629. [8] J.I. Inokuchi, K.I. Inamori, K. Kabayama, M. Nagafuku, S. Uemura, S. Go, et al., Biology of gm3 ganglioside, Prog. Mol. Biol. Transl. Sci. 156 (2018) 151–195. [9] T. Yamashita, A. Hashiramoto, M. Haluzik, H. Mizukami, S. Beck, A. Norton, et al., Enhanced insulin sensitivity in mice lacking ganglioside gm3, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 3445–3449. [10] J.I. Inokuchi, S. Go, M. Yoshikawa, K. Strauss, Gangliosides and hearing, BBA 2017 (1861) 2485–2493. [11] M. Yoshikawa, S. Go, S. Suzuki, A. Suzuki, Y. Katori, T. Morlet, et al., Ganglioside gm3 is essential for the structural integrity and function of cochlear hair cells, Hum. Mol. Genet. 24 (2015) 2796–2807. [12] A. Boukhris, R. Schule, J.L. Loureiro, C.M. Lourenco, E. Mundwiller, M.A. Gonzalez, et al., Alteration of ganglioside biosynthesis responsible for complex hereditary spastic paraplegia, Am. J. Hum. Genet. 93 (2013) 118–123. [13] G.V. Harlalka, A. Lehman, B. Chioza, E.L. Baple, R. Maroofian, H. Cross, et al., Mutations in b4galnt1 (gm2 synthase) underlie a new disorder of ganglioside biosynthesis, Brain 136 (2013) 3618–3624.

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Please cite this article as: K. Aoki, A. D. Heaps, K. A. Strauss et al., Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency, Clinical Mass Spectrometry, https://doi.org/10.1016/j.clinms.2019.03.001