Phospholipidomics of Human Blood Microparticles - Analytical

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Phospholipidomics of human blood microparticles Ilario Losito, Raffaele Patruno, Elena Conte, Tommaso R.I. Cataldi, Francesco M. Megli, and Francesco Palmisano Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac400829r • Publication Date (Web): 07 Jun 2013 Downloaded from http://pubs.acs.org on June 16, 2013

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Analytical Chemistry Short title: Phospholipidomics of human blood microparticles

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Phospholipidomics of human blood microparticles I. Losito1,2, *, R. Patruno1, E. Conte3,§, T.R.I. Cataldi1,2, F.M. Megli3,4, F. Palmisano1,2

1

Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro”, Via E. Orabona 4,

70126 Bari, Italy 2

Centro Interdipartimentale SMART, Università degli Studi di Bari “Aldo Moro”, Via E.

Orabona 4, 70126 Bari, Italy 3

Dipartimento di Biochimica e Biologia Molecolare “E. Quagliariello”, Università degli

Studi di Bari “Aldo Moro”, Via E. Orabona 4, 70126 Bari, Italy 4

Centro di Studio sui Mitocondri e Metabolismo Energetico, Consiglio Nazionale delle

Ricerche, Via E. Orabona, 4, 70126 Bari, Italy §

Current address: Dipartimento Farmaco-Biologico, Università degli Studi di Bari “Aldo

Moro”, Via E. Orabona 4, 70126 Bari, Italy

* Corresponding Author: e-mail: [email protected] Phone: 0039 080 5442020 Fax: 0039 080 5442092

ACS Paragon Plus Environment

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Analytical Chemistry

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Short title: Phospholipidomics of human blood microparticles

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Abstract The phospholipidome of blood microparticles (MPs) obtained from platelet-rich plasma of healthy individuals was characterized by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). The HILIC separation, performed on a silica stationary phase using an acetonitrile/methanol gradient, enabled the separation of several phospholipids (PL) classes, viz. phosphatidylcholines (PCs), -ethanolamines (PEs), -serines (PSs), -inositoles (PIs), sphyngomielins (SMs) and lyso forms of PCs and PEs. Structural characterization of species belonging to each class was performed by MS/MS measurements, either in positive or negative ion mode. The set of 131

phospholipids

(including

regioisomers)

here

identified

represents

the

most

comprehensive phospholipidomic characterization reported for human MPs. Although the phospholipidome composition of MPs and platelets, collected from the same donors, was found to be qualitatively the same, quantitative differences were evidenced for lyso-PCs, that appear to be significantly more abundant in MPs.


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Introduction Cellular microparticles (MPs) are membrane vesicles having sub-micrometric dimensions released from virtually every cell type. Although MPs have been assimilated to cellular debris for a long time after their discovery1, they are currently being reconsidered for their role in processes like cellular stimulation, activation and degeneration/apoptosis.2,3 Specifically, blood MPs, i.e. microparticles released from erythrocytes, white blood cells, platelets and the endothelial lining of blood vessels (see Ref. 4 and references cited therein), have been recognized as important actors either in the normal hemostatic response to vascular injury or in several human pathological conditions, including haemolysis, sickle cell anemia, inflammation,

atherosclerosis,

type-2

diabetes

mellitus,

diabetic

nephropathy

and

cardiovascular diseases.5-12 Several efforts have been devoted to blood MP counting, dimensional analysis and evaluation of cellular origin4,9,13-18. In particular, diameters ranging from 1 µm down to 10 nm have been reported, the lowest values being measured using atomic force microscopy.17,18 At a more sophisticated level of information, the antigenic determinant of blood MPs has been studied with the aim to identify their cellular origin; indeed all MPs bear specific proteins on their outer surface, representing a signature of the cells they are generated from. For instance, glycophorin A (CD235a), a protein uniquely expressed on the erythrocytes membrane, was found on the surface of erythrocyte-derived MPs, whereas CD61, CD41 and CD42 proteins are specific of platelet-derived MPs (PMP).4 Antibody capture-based enzyme linked immunosorbent assays (ELISA)19 or flow cytometry20 have been then exploited for the antigenic characterization of cell MPs. Gold-labeled specific antibodies have been also used to tag blood MPs for electron microscopy experiments.21 It is worth of mention that plateletderived MPs are, by far, the most important sub-population, representing up to 70%, whereas white and red blood cell-derived MPs account only for 3 and 8%, respectively.22


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A quite exhaustive proteomic characterization of MPs has been performed during the last decade.23-26 Jin et al. reported a three-fold higher number of protein-related spots for plasma MPs compared to whole plasma23; moreover, 30 out of 83 identified proteins were associated solely to MPs. Likewise, a remarkable number of new proteins, not observed in previous studies dealing with the platelet proteome, were reported by Garcia and coworkers on PMPs obtained through artificial activation.24 Lipidomics of blood MPs has received a much lower attention than proteomics. Limited information on the distribution of phospholipid classes, obtained using high performance thin layer chromatography, have been reported for native human blood MPs25 and

for

MPs

obtained

through

platelets

activation

(PMPs).26

In

both

cases,

phosphatidylcholines (PCs), sphyngomielins (SMs) and phosphatidylethanolamines (PEs) appeared as the most abundant phospholipid classes, their relative amounts being, however, quite different (PCs were more abundant in native MPs compared to PMPs). To the best of our knowledge, a comprehensive chemical characterization of the phospholipidome of human blood MPs has not been reported yet; for this reason a systematic investigation has been undertaken in our laboratories. A preliminary comparison between the separation efficiency offered by hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) clearly indicated that HILIC, coupled to electrospray ionization mass spectrometry (ESI-MS) in tandem mode configuration (MS/MS), was the method of choice for PLs identification, including the assessment of their regiochemistry (i.e., acyl/alk(en)yl chains location on the sn1 and sn2 positions of glycerol). Here, the results of this investigation are described, along with a qualitative comparison between the PL profiles of human blood MPs and platelets (the main source of blood MPs).


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Experimental Solvents and chemicals. Water, methanol and acetonitrile (LC-MS grade), chloroform (HPLC-grade), ammonium acetate, tris-(2-hydroxy-methyl)-aminomethane hydrochloride (TRIS-HCl) and ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma-Aldrich (Milan, Italy). Phospholipid standards (all ≥ 99% purity) were purchased from Avanti Polar Lipids (Alabaster, AL, US); the complete list is reported in the Supporting Information.

Sample preparation. Platelet-rich plasma (PRP) was collected by plateletpheresis from 40 – 45 years aged healthy donors and processed within 48 hours at maximum. During this time the sealed PRP storage bags were kept in constant horizontal agitation at room temperature (20-24 °C). A pellet containing platelets and a microparticle-enriched supernatant were obtained from a 20 mL aliquot of PRP by centrifugation at 1,500 g for 15 min at 4 °C (a temperature deliberately chosen to minimize any possible alteration of phospholipids). Note that the centrifugation step is typically performed at room temperature27,28 to minimize possible cold-induced activation effects such as those observed for some haemostasis factors in citrated blood stored at 4°C for ca. 4 hours29. However, cold-induced phenomena, if present, can be reasonably assumed to be negligible considering the time scale (15 min) of the PRP centrifugation step. The platelet-enriched pellet was re-suspended in 3 mL of a wash buffer (pH 7.4) containing NaCl (154 mmol/L), TRIS-HCl (10 mmol/L) and EDTA (1 mmol/L) and centrifuged at 1500 g for 10 min at 4 °C. The blood microparticle-enriched supernatant was subjected to ultracentrifugation at 19,000 g for 60 min at 4 °C, in order to form a pellet of blood MPs. The supernatant was removed carefully and the pellet was re-suspended in 1 mL of wash buffer at pH 7.4. In order to evaluate the presence of cellular contaminants a suspension of MPs was subjected to Transmission Electron Microscopy (TEM) measurements


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after negative staining by phosphotungstic acid30 as detailed in the Supporting Information. TEM images (see Figures S-1 A and B) showed the presence of MPs with diameters ranging between a few tens and a few hundreds of nanometers. MPs aggregates (ca. 1-2 µm sized) were occasionally observed, whereas no evidence was obtained for the presence of residual platelets or cellular debris. Extraction of PLs from platelets and blood MPs, isolated as described before, was performed according to the Bligh and Dyer protocol.31 Extracted PLs were stored in 1 mL of chloroform and assayed for their phosphorous content32 in order to estimate PLs concentration (assuming one P atom per PL molecule). An aliquot of the chloroform extract, containing 0.51.0 µmoles of phosphorous, was withdrawn, dried under a nitrogen flow, to minimize airinduced oxidation processes, and then sealed in glass ampoules, which were stored at -27 °C.

LC-MS instrumentation and operating conditions. A Dionex P680 (Dionex, Sunnyvale, CA, USA) liquid chromatography pump, connected to a Supelco-Ascentis (Supelco, Bellefonte, PA, USA) silica column (15 cm × 2.1 mm, packed with 3 µm particles), was used for HILIC separations of PLs. The column effluent (flow rate: 0.3 mL/min) was transferred, without splitting, to the electrospray interface of an LCQ 3D ion trap mass spectrometer (Thermo Scientific, West Palm Beach, FL, USA). The following binary elution program, based on acetonitrile (solvent A) and methanol (solvent B), both containing 10% water (v/v) and 2.5 mmol/L of ammonium acetate, was adopted: 0-6 min, isocratic at 9% of solvent B; 611 min, linear to 36% B; 11-20 min, isocratic; 20-25 min, back to initial composition, followed by 20 min equilibration time. For comparative purposes, a RPLC separation was also accomplished on a PL extract of blood MPs using a SupelcosilTM (Supelco, Bellefonte, PA) C18 column (25 cm × 2.1 mm, packed with 5 µm particles) at a 0.2 mL/min flow rate. The following binary elution program, based on water (solvent A) and methanol (solvent B),


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both containing 2.5 mmol/L of ammonium acetate, was adopted: 0-50 min linear from 80 to 100 % (v/v) of solvent B; 50-80 min isocratic, 80-85 min back to the initial composition followed by 20 min equilibration time. Unless otherwise specified the injection volume was always 20 µL. Just before LC-MS analysis the phospholipid extracts were reconstituted in a methanol/chloroform 1:1 (v/v) mixture to a final P concentration of 2 nmol/µL and the resulting solutions rapidly transferred into glass vials with screw caps and maintained under a nitrogen atmosphere at -27 °C, between replicated analyses on the same sample. MS full scan acquisitions were performed, either in positive or negative ion mode, in the m/z range 50-2000. The following values were adopted for the ESI interface and ion optics parameters: sheath gas flow rate, 60 (a.u.); auxiliary gas flow rate, 0 (a.u.); spray voltage, 6 kV (positive polarity)/-4.5 kV (negative polarity); capillary temperature, 190 °C; capillary voltage, 15 V/-45 V; tube lens offset, 10 V/-5 V; octapole 1 offset, -2.5/1.5 V; lens voltage, -16/34 V; octapole 2 offset, -5.5/6.5 V; octapole RF amplitude, 400 Vp-p. Subsequently, MS/MS acquisitions were performed on targeted precursor ions. Only the M and M+1 isotopologues of each precursor ion were isolated before fragmentation, using a 2 m/z units-wide window centered on the m/z ratio of the M isotopologue. The M+2 isotopologue was always excluded from isolation and fragmentation to avoid spectral interferences, since the presence of partially co-eluting species having 2 m/z units-spaced m/z ratios was very common in the analyzed samples. Preliminary measurements on representative PL standards showed that 35% collisional energy, corresponding to a 1.75 V peak-to-peak value for the excitation voltage applied to the 3D ion trap end-caps, enable an almost complete fragmentation of the precursor ions. The Xcalibur software (Thermo Scientific) was used to control the acquisitions and to perform the initial elaboration of data.


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Results and discussion Class-related HILIC separation of MP phospholipids A silica stationary phase operating under HILIC conditions was adopted to separate mixtures of PLs extracted from blood MPs or platelets obtained from the same donors. The relevant total ion current (TIC) chromatograms are compared in Figures 1A and 1B. In accordance with other HILIC-based analyses of PL mixtures33-38 distinct bands (corresponding to different classes of PLs) could be observed, since retention was mainly dictated by the polarity of the phospholipid heads. This behavior is strikingly different from the one obtained by Reverse Phase Chromatography (RPC). As evidenced by Figure 1C, referred to the same blood MP extract of Figure 1A, in spite of the remarkably longer run time a single, unresolved wide band was obtained. The well-known dependence of RPC retention on the hydrophobicity of the side chains prevents PLs from being separated into classes. When complex phospholipidomes, rather then mixtures of PLs from a specific class, are analyzed, this feature leads to a significant lack of chromatographic resolution and, at the same time, increases the risk of coelution of isobaric species belonging to different classes. Actually, the RPC resolution is impaired also by the inherent peak width of each PL, which is significantly larger than that observed using HILIC. For the sake of example, the HILIC eXtracted Ion Current (XIC) chromatogram obtained for the m/z ratio 703.6, afterwards assigned to a sphyngomielin using MS/MS data, is reported in Figure S-2 of the Supporting Information and compared with the RPC one (see the inset). RPC-ESI-MS separations performed on a palmitoyl-linoleyl-phosphatidyl-choline standard, showed a systematic narrowing of the peak width at the baseline, from ca. 10 to 2 min, when the concentration was lowered from 2.5 to 0.025 nmol/µL, suggesting the occurrence of aggregation phenomena in the polar mobile phase. Such aggregates might exhibit a slightly different retention on the C18 stationary phase, due to the different overall number of hydrophobic side chains, thus leading to


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significant band broadening. Such aggregates are disrupted, either in the ESI source or into the heated transfer capillary of the mass spectrometer, thus monomeric ions can be detected. Bands observed in the chromatograms of Figures 1A and 1B likely originate from the co-elution of PLs belonging to a given class, as suggested by mass spectra averaged over each HILIC band (see Figure 2 for an example). Clusters of peaks spaced by 28 m/z units, (reflecting differences of two CH2 units in the PL side acyl chains), are evident in the spectrum. Additionally, although less intense, clusters of peaks spaced by 14 m/z units are observed, indicative of the occurrence of either PLs species differing by one CH2 unit on the acyl chains or of 1,2-di-acyl- and 1-alkyl-2-acyl PLs bearing the same total number of carbon atoms. The prevalence of the 28 m/z units-spaced species is not surprising, since acyl-chains with an odd number of carbon atoms are not very common in natural PLs39 and 1-alkyl-2acyl-PCs are usually much less abundant than di-acyl-PCs.40 Interestingly, each cluster in the Figure 2 spectrum is clearly characterized by the presence of several 2 m/z units-spaced peaks, a feature related to the presence of PL species belonging to the same class, bearing the same total number of carbon atoms on their side chains but differing for the presence of just a C=C double bond on the latter. On this basis, it is apparent that a snapshot of an entire PL class can be retrieved from mass spectra underlying HILIC bands, a significant advantage with respect to RPC separations, at least when complex phospholipidomes are analyzed. A confirmation of the successful class-based PLs separation achievable by HILIC is shown by Figure 3, where eXtracted Ion Current (XIC) chromatograms for selected species belonging to the PC, SM and lyso-PC classes, obtained from the TIC trace in Figure 1B are compared. Representative XIC traces relevant to other classes of PLs detectable either as positive ions, i.e., PE, 1-alkenyl-2-acyl-PE (alias plasmenyl-PE or pPE), lyso-PE and PS, or as negative ions, i.e. PIs, are shown in Figures S-3 and S-4, respectively. The presence of isobaric/isomeric species is clearly suggested by some of the XIC traces in Figures 3 and S-3.


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However, possible ambiguities in the final identification of each peak could be subsequently removed using MS/MS or MS3 measurements (vide infra). Typically, the identification procedure started with a preliminary search on the freely accessible LipidMaps database (www.lipidmaps.org), targeted on all the m/z ratios selected for ion current extraction. Candidate species were thus retrieved for almost all PL classes, the only exceptions being PSs and lyso-PEs, whose bands cannot be observed distinctly in the HILIC-MS TIC chromatogram. Indeed, as shown in Figure 1A and, more specifically, in Figure S-3, PSs eluted in a 2.5-4 min retention time interval, i.e. on the tail of the early eluting band. In the case of lyso-PEs a band could not be observed in the TIC trace due to their low abundance. Consequently, ion current extractions were initially performed on m/z ratios calculated for PSs and lyso-PEs characterized by acyl chain combinations usually found in other PLs or lyso-PLs (e.g. 16:0/18:2, 18:0/18:2, 16:0/20:4, 16:0/0:0, 18:0/0:0, etc.). Peaks observed in the resulting XIC traces were then used to locate the elution intervals for PS and lyso-PE classes and the m/z ratios of further species potentially belonging to the two classes were retrieved from the MS spectra averaged over those intervals. Finally, the search for correlated PLs was performed on the LipidMaps database. It is worth noting that a crosscheck of the set of m/z ratios finally obtained for each sample was performed also using the IntelliXtract routine, included in the MS-Manager software package (ACD/Labs, Toronto, Canada). The IntelliXtract software performs an automatic extraction of chromatographic components in complex LC/MS datasets, provided input information like mass accuracy and intensity threshold are fixed by the user. In the present case an excellent match between the set of m/z ratios retrieved after manual extraction of ion currents and that arising from the IntelliXtract-based elaboration of LC-MS data was obtained.


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MS/MS- or MS3–based identification of blood MP phospholipids MS2 and MS3 experiments were performed both in positive (for all PLs but PIs) and negative polarity in order to choose the best ionization mode in terms of regiochemical information retrievable. Positive ion MS/MS allowed the unambiguous identification of all PL classes but PCs and PIs, just on the base of diagnostic polar head-related fragmentations41-43. Indeed, neutral losses of phosphoethanolamine (C2H8NO4P, nominal mass 141 Da) and phosphoserine (C3H8NO6P, nominal mass 185 Da) were observed, respectively, for PEs, plasmanyl- (oPE), plasmenyl-PEs (pPE) and lyso-PEs and for PSs. In the case of PCs, lyso-PCs and SMs the observation of the diagnostic phosphocholine ion, at m/z 184, was usually prevented by the low mass cut off automatically set for 3D ion trap MS/MS spectra. Nonetheless, the spontaneous in-source generation of the phosphocholine ion enabled the recognition of HILIC bands related to PCs, SMs and lyso-PCs in the XIC traces retrieved from LC-MS TIC chromatograms for the 184 m/z ratio. Moreover, both SMs and lyso-PCs were characterized by a water loss ([M+H-H2O]+) and SMs showed an additional trimethylamine loss from the choline head ([M+H-H2O-(CH3)3N]+) when MS/MS was accomplished. Positive ion MS/MS measurements provided regiochemical information only in the case of 1-alkyl-2-acyl- (oPE) or 1-alkenyl-2-acyl-phosphatidylethanolamines (pPE) and of lyso-PLs. In particular, the fragmentation pattern observed for oPE/pPEs, exemplified in Figure S-5 for standard PE(P-18:0/18:1) and described in general terms in Table 2, was in excellent agreement with those reported by Berry and Murphy44 and enabled an easy identification of the saturated or mono/polyunsaturated chain linked, by an ether bond, to the sn1 oxygen of glycerol and of the acyl chain linked to the sn2 oxygen. In the case of lyso-PCs and lyso-PEs, the ratio between the abundance of the [M+HH2O]+ peak and that of the phosphocholine ion (m/z 184) or of product ions arising from phosphoethanolamine/ethanolamine loss, respectively, was exploited to locate the residual


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acyl chain. In the case of lyso-PCs a ratio higher than 1 has been reported as diagnostic for a sn1 location (and viceversa).45 Such a pattern was confirmed experimentally by the MS/MS spectrum obtained with our MS instrument for standard PC(16:0/0:0), reported in Figure S-5. The [M+H-H2O]+/[phosphocholine]+ abundance ratios observed in the MS/MS spectra of lyso-PCs detected in platelet or MP extracts suggest the presence of two distinct groups of species. For the first group the ratio was always higher than 1.3, whereas in the second it never exceeded 0.5. Such a difference made it reasonable to identify the corresponding lysoPCs as PC(n:m/0:0) and PC(0:0/n:m), respectively (with n representing the total number of carbon atoms and m the number of C=C bonds along the side chains). Interestingly, two couples of regioisomeric lyso-PCs were also identified (vide infra). As for lyso-PEs, previous positive ion MS/MS measurements made by Lee et alt. on standards had shown only an increase of the [M+H-H2O]+/ [M+H-phosphoethanolamine]+ abundance ratio when passing from PE(0:0/14:0) to PE(14:0/0:0), yet the ratio never exceed the unity45. On the contrary a ratio higher than 1.3 was observed in the MS/MS spectrum of standard PE(16:0/0:0) using our spectrometer (see Figure S-5). When lyso-PEs present in platelet/MP extracts were considered, the ratio was found to be comprised between 1.3 and 1.4 for six species and to be always lower than 0.5 for as many species. The MS/MS spectrum of standard PE(16:0/0:0) and the analogy with the behavior of lyso-PCs, led to assign the two groups of lyso-PEs as PE(n:m/0:0) and PE(0:0/n:m), respectively. Two couples of regioisomeric species were identified also for this class (vide infra). Negative ion MS/MS measurements were made on different precursor ions according to the class of PLs. As shown in Figure S-6 A, reporting the MS spectrum of PC(18:0/18:1), three diagnostic product ions were detected for PLs having phosphocholine as the polar head, namely: [M+AcO]-, [M+H+AcO-CH3]- and [M-CH3]-. Here M represents the zwitterionic phospholipid and AcO the acetate anion, likely arising from the HILIC mobile phase.


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Conversely from what observed using a triple quadrupole (see Fig. 8 in Ref. 43), the 3D ion trap MS/MS fragmentation of [M+AcO]- and [M+H+AcO-CH3]- ions normally led only to [M-CH3]- in the present case (see Figure S-6 A). This behavior was also established for standard SMs and lyso-PCs and confirmed for all the PLs containing a phosphocholine moiety occurring in MP or platelet lipid extracts. Another interesting dissimilarity between triple quadrupole and 3D-ion trap, already described in the literature43, emerged from the fragmentation of the [M-CH3]- ion, as shown in Figure S-6 B for standard PC(18:0/18:1). Indeed, the loss of acyl chains as ketenes (m/z 508.4 and 506.4) was found to prevail over their loss as fatty acids (m/z 490.4 and 488.4) or the generation of fatty acid anions (m/z 281.5 and 283.5), that were dominating in triple quadrupole-based MS/MS spectra. Nonetheless, the ketene loss from the sn2 position was clearly prevailing over that occurring from the sn1 position, as observed previously43, thus providing a key to assess the regiochemistry of all the PCs detected in blood MP extracts (see Table 2). As exemplified in Figure S-7, the fragmentation of [M-CH3]- ions arising from SMs and lyso-PCs led to the diagnostic loss of the single acyl chain as a ketene; additionally, its lost as a fatty acid and the generation of the corresponding fatty acid anion were observed in the case of lyso-PCs. Since no adducts with acetate or related ions were found in the tandem MS spectra of PEs and lyso-PEs, their deprotonated molecules, [M-H]-, were chosen as precursor ions. The fragmentation patterns observed for these classes of lipids were similar to those observed for PCs and lyso-PCs, respectively, as exemplified in Figures S-8 and S-9, respectively, and as described in Table 2. Concerning phosphatidylserines (PSs), tandem MS on [M-H]- ions was unable to provide a clear information on the acyl chain, the main product ion being that arising from the serine loss, i.e. the anion of a phosphatidic acid, as shown in Figure S-10. However, MS3 experiments on this ion gave rise to diagnostic fragments, with acyl chains preferentially lost


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as fatty acids, rather than ketenes (see Figure S-10), and mainly from the sn2 position, in accordance with the general model reported for the fragmentation of ions, like those of PSs, exhibiting gas phase acidity.43 As shown in the MS/MS spectrum reported in Figure S-11, confirming previous investigations46, the predominance of fatty acid loss was observed also for the only standard PI available, having two identical side chains. The previously reported prevalence of fatty acid loss from the sn2 position46, although not verifiable experimentally in the present case, was exploited for the assignment of the regiochemistry of PIs detected in blood MP and platelet extracts, all having different side chains.

Phospholipid profiles of human blood MPs and platelets. A summary of all the PLs identified both for blood MPs and platelets, itemized into PC, SM, lyso-PC, PE, pPE/oPE, lyso-PE, PS and PI classes, is provided in Table 1. A label indicating the relative abundance observed for the peak of each species in the MS spectrum of the corresponding class was added in parentheses: h (high, > 80%); m (medium, 20-80%); l (low, < 20%). For all the PLs but the lyso forms the two side chains are indicated in the table, in the sn1/sn2 order, with the usual notation, i.e. “number of carbon atoms:number of C=C bonds”. The missing chain of lyso-PLs is represented by the 0:0 notation. In the case of plasmenyl/plasmanyl-phospholipids (oPLs/pPLs) the side chain linked to glycerol through an ether bond (sn-1 position) is indicated with a P-/O- followed by the usual chain notation. As described in the previous section, the regiochemistry of the PL species could be ordinarily assessed using MS/MS or MS3 data, the only exception being represented by some plasmanyl/plasmenyl-PCs, whose concentration was too low to enable a reliable MS/MS characterization (only the overall composition of the two chains, one alk(en)ylic and the other acylic, is thus provided in Table 1). It is worth noting that none of the fragmentations discussed above could provide information on the location and configuration of the C=C


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double bonds along the alkyl or acyl chains of PLs. Nonetheless, they can be reasonably assumed to be the same as those usually reported in the literature, e.g. 9Z for 18:1 chains; 9Z,12Z for 18:2; 5Z,8Z,11Z,14Z for 20:4, and so on.39 To the best of our knowledge, the 131 PLs listed in Table 1 represent the first holistic characterization ever reported for the blood MP phospholipidome and, additionally, the most extended phospholipidomic profile obtained for platelets. It is important to point our that our choice of focusing on a qualitative comparison between the phospholipidomes of blood MPs and platelets arose from the fact, already discussed in the Introduction, that platelets are, by far, the main sources of blood MPs. On the other hand, since their separation from MPs by centrifugation is almost impossible4, the presence in our MP samples (and hence the contribution to the observed phospholipidome) of the small membrane vesicles (diameter 40-100 nm) found in plasma and usually termed exosomes cannot be excluded. On the basis of TEM images obtained from the investigated blood MP samples, the contribution due to the larger vesicles (i.e., > 1.5 µm) known as apoptotic bodies4 and to platelets, escaping the first centrifugation step performed on plateletrich plasma, can be considered negligible. Recently, two MS-based characterizations of platelet PLs have been reported by Leidl et al.47 and Ruebsaamen et al.48. In both cases the identification of PLs was focused on the overall composition of the side chains for each species, yet an interesting comparison can be made with the findings of the present paper. Species with a total number of carbon atoms corresponding to each of the even numbers comprised between 32 and 40, for PCs, and between 34 and 40, for PEs, and characterized by a maximum of six C=C double bonds were reported previously.47 The agreement with the present study is apparent from Table 1, although PLs characterized by a higher degree of unsaturation were detected both in blood MPs and platelets, with the total number of C=C bonds raising up to 8 for some species. The


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higher incidence of unsaturated chains in the PLs of blood MP and platelet extracts was also observed for plasmanyl-PC and, more clearly, for plasmenyl-PE species. For the latter class 15 further species, usually characterized by O-linked chains longer than those indicated in the previous studies, were identified. As for SMs, Leidl et al.47 assumed that the alkyl chain (including the glycerol skeleton in the case of SMs) was always a 18:1 and inferred the nature of the acyl chain accordingly. Our MS/MS-based specific assignment of the two chains is in agreement with such an assumption in most cases (see the SM section in Table 1), although SMs having a 16:1 or a 17:1 alkyl chain were also identified both in blood MPs and platelets. A general accordance with the previous study was also found about the overall composition of acyl chains for platelet PIs, PSs and lyso-PCs, whereas lyso-PEs were reported here for the first time. Both classes of lyso-PLs detected in MPs and platelets deserve a special comment. Indeed, relatively short chains (e.g. 16 and 18 carbon atoms) are mainly located on the sn1 position of glycerol, whereas long, highly unsaturated chains (having 20 and 22 carbon atoms and 3 to 6 C=C bonds) are usually found on the sn2 position. Both the possible regioisomers for each lyso-PL were detected only in a few cases, namely two for each class. These results are in agreement, at least in terms of acyl chain identity, with those reported in a recent investigation on lyso-PLs extracted from human plasma45, although a higher number of couples of regioisomeric lyso-PLs was identified in that matrix. Last, but most importantly, a key role seems to be played by lyso-PCs in the differentiation between blood MP and platelet phospholipidomes, as suggested by the comparison between HILIC-MS TIC profiles obtained from PL extracts of blood MPs and platelets (see Figures 1A and B). The lyso-PC band, eluting at approximately 16.5-18.5 min, is relatively more abundant and is changed in shape, thus suggesting a different quantitative distribution of species, in the case of blood MPs. The same behavior was observed in all the


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six couples of MP/platelet samples examined during the present study. The incidence observed for lyso-PCs in blood MPs and platelets is also in accordance, at least from a qualitative point of view, with that inferred from previous data based on high performance thin

layer

chromatography

(HPTLC)

separations

followed

by

photodensitometric

measurements25,26. Indeed, as reported by Birò et al.26, the abundance of lyso-PCs was found to be very low both in the phospholipid extracts of plasma, granule and intracellular membranes of platelets and in those of MPs obtained by different platelet activations. On the contrary, HPTLC data reported by Weerheim et al.25 suggested the presence of lyso-PCs (and also, in one case, of other lyso-PLs) in the PL extracts of plasma MPs obtained from two healthy donors. Moreover, both investigations showed the general prevalence of PCs, SMs and PEs in the phospholipidomes of MPs (native or activated) and platelets, in accordance with the present data (see Figures 1A and 1B). Since lyso-PLs are known to induce a significant physical perturbation on PL-based membranes49, their peculiar abundance in blood MPs is rather surprising in terms of stability. Further differences in the distribution of species were suggested by qualitative comparisons of ESI-MS spectra averaged under the same band for the MPs and platelets studied during this investigation. A quantitative evaluation of such distributions could then provide relevant information on the relationship existing between the phospholipidome of blood MPs and platelets.


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Conclusions HILIC combined with single, tandem and sequential (MS3) 3D-ion trap mass spectrometry was successfully applied to investigate the phospholipidome of human blood MPs. Along with the recognition of the main PL classes, a detailed profiling of each class, leading to a set of 131 PL species, was achieved. The same set of PLs was identified in extracts obtained from platelets of the same donors. These results represent, to the best of our knowledge, the most complete characterization currently available for the phospholipidome of human blood MPs and platelets. Several new PL species, most of which containing longer and more unsaturated side chains than those reported in a previous study on platelets, were identified in both sample types. A higher abundance of lyso-PCs, which are known to be membrane-destabilizing species, was observed in blood MPs extracts, compared with extracts obtained from platelets. Further work is in progress to analyze such differences in more detail and to find a possible relationship with the relevant properties of blood MPs.

Acknowledgements Financial support from the Italian Ministero per l'Istruzione, l'Università e la Ricerca (MIUR), through the research project PRIN 2009KW27KE_003, is gratefully acknowledged. Drs. Francesco Cucci, Egidio Conte and Giacomo Bellomo (Di Suma-Perrino Hospital, Brindisi, Italy) are acknowledged for kindly donating platelet-rich plasma samples. Dr. Adriana Trapani (Department of Pharmaceutical Chemistry, University of Bari “Aldo Moro”) and Prof. Nicola Cioffi (Department of Chemistry, University of Bari “Aldo Moro”) are acknowledged for the preparation of MP samples for TEM measurements and for the acquisition of TEM images, respectively.


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(21) Aras, O.; Shet, A.R.; Bach, R.R.; Hysjulien, J.L.; Slungaard, A. Hebbel, R.P.; Escolar, G.; Jilma, B.; Key, N.S. Blood 2004, 103, 4545-4553. (22) Boselli, D.; Brambilla, M.; Tremoli, E.; Manganaro, D.; Camera, M. Blood Transfusion 2012, 10-Supp. 4, S112. (23) Jin, M.; Drwal, G.; Bourgeois, T.; Saltz, J.; Wu, H.M. Proteomics 2005, 5, 1940-1952. (24) Garcia, B.A., Smalley, D.M.; Cho, H.J.; Shabanowitz, J.; Ley, K.; Hunt, D.F. J. Proteome Res. 2005, 4, 1516-1521. (25) Weerheim, A.M.; Kolb, A.M.; Sturk, A.; Nieuwland, R. Anal. Biochem. 2002, 302, 191-198. (26) Biró, É.; Akkerman, J.W.N.; Hoek, F.J.; Gorter, G.; Pronk, L.M.; Sturk, A.; Nieuwland, R. J. Thromb. Haemost. 2005, 3, 2754-2763. (27) Yuana, Y.; Bertina, R.M.; Osanto, S. Thromb. Haemost. 2011, 3, 396–408. (28) Dey-Hazra, E.; Hertel, B.; Kirsch, T.; Woywodt, A.; Lovric, S.; Haller, H.; Haubitz, M.; Erdbruegger, U. Vasc. Health Risk Manag. 2010, 6, 1125-1133. (29) Favaloro, E.J.; Soltani, S.; McDonald, J. Am. J. Clin. Pathol. 2004; 122, 686-692. (30) Yashroy, R.C. J. Biosci. 1990, 15, 93-98. (31) Bligh, E.G.; Dyer, W.J. Can. J. Biochem. Phys. 1959, 37, 911-917. (32) Nakamura, G.R. Anal. Chem. 1952, 24, 1372. (33) Desoubzdanne, D.; Claparols, C.; Martins-Froment, N.; Zedde, C.; Balayssac, S.; Gilard, V.; Tercé, F.; Martino, R.; Malet-Martino, M. Anal. Bioanal. Chem. 2010, 398, 2723-2730. (34) Schwalbe-Herrmann, M.; Willmann, J.; Leibfritz, D. J. Chromatogr. A 2010, 1217, 5179-5183. (35) Zheng, L.; T’Kind, R.; Decuypere, S.; von Freyend, S.J.; Coombs, G.H.; Watson, D.G. Rapid Commun. Mass Spectrom. 2010, 24, 2074-2082. (36) Okazaki, Y.; Kamide, Y.; Hirai, M.Y.; Saito, K. Metabolomics 2013, 9, S121-S131. (37) Xiong, Y.; Zhao, Y.Y.; Goruk, S.; Oilund, K.; Field, C.J.; Jacobs, R.L.; Curtis, J.M. J. Chromatogr. B 2012, 911, 170-179. (38) Zhu, C., Dane, A.; Spijksma, G.; Wang, M.; van der Greef, J.; Luo, G.; Hankemeier, T.; Vreeken R.J. J. Chromatogr. A 2012, 1220, 26-34. (39) Hanahan, D.J. A guide to phospholipid chemistry, Oxford University Press: New York, 1997, Chapter 1, pp. 3. (40) Sugiura, T.; Waku, K. In Platelet-activating factor and related lipid mediators; Snyder, F, Ed.; Plenum Press: New York, 1987; pp 55.


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(41) Pulfer, M.; Murphy, R.C. Mass Spectrom. Rev. 2003, 22, 332-364. (42) Han, X.; Gross, R.W. Mass Spectrom. Rev. 2005, 24, 367-412. (43) Hsu, F.F.; Turk, J. J. Chromatogr. B 2009, 877, 2673-2695. (44) Berry, K.A.Z.; Murphy, R.C. J. Am. Soc. Mass Spectrom. 2004, 15, 1499-1508. (45) Lee, J.Y., Min, H.K.; Moon, M.H. Anal. Bioanal. Chem. 2011, 400, 2953-2961. (46) Hsu, F.F.; Turk, J. J. Am. Soc. Mass Spectrom. 2000, 11, 986-999. (47) Leidl, K.; Liebisch, G.; Richter, D.; Schmitz, G. BBA-Mol. Cell Biol. L. 2008, 1781, 655-664. (48) Ruebsaamen, K.; Liebisch, G.; Boettcher, A.; Schmitz, G. Transfusion 2010, 50, 16651676. (49) Colles, S.M., Chisolm, G.M. J. Lipid Res. 2000, 41, 1188–1198.


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Captions to Figures Figure 1. HILIC-ESI-MS total ion current (TIC) chromatograms relevant to the phospholipid extracts of A) blood MPs and B) platelets obtained from the same healthy donor. For the sake of comparison the RPC-ESI-MS TIC trace relevant to the blood MP extract is shown in panel C. The assignment of HILIC bands to PL classes was performed with the help of MS/MS data; see text for details. Legend for PL classes: PS (phosphatidylserines), PE (1,2-diacylphosphatidylethanolamines), pPE (1-alkenyl- 2-acyl-phosphatidylethanolamines), PC (1,2diacyl-phosphatidylcholines),

oPC

(1-alkyl-2-acyl-phosphatidylcholines)

and

SM

(sphyngomielins).

Figure 2. ESI-MS spectrum averaged over the band observed for PCs in the HILIC-ESI-MS TIC chromatogram shown in Figure 1A. The distinctive 28 m/z units spacing observed between peak clusters detected in the two spectra is emphasized.

Figure 3. eXtracted Ion Current (XIC) chromatograms obtained for representative PCs, SMs and lyso-PCs after the positive ion HILIC-ESI-MS analysis of the phospholipid extract of a blood MPs sample. The peak actually referred to SM(d18:1/24:1) is marked by an arrow.


22

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Table 1. Overview of PLs identified by HILIC-ESI-MS and MSn (n = 2,3) measurements in the lipid extracts of blood microparticles and platelets withdrawn from healthy donors. Class PC/oPC 27 species

SM 20 species

Species a O-32:0 (l) 16:1/ 16:1 (l)

m/z ratio 720.5 730.6

16:0/ 16:1 (l) 16:0/ 16:0 (m) O-34:2 (l) O-34:1 (l) 16:0/ 18:3 (l) 16:0/ 18:2 (h) 16:0/ 18:1 (h)

732.6 734.5 744.5 746.7 756.5 758.5 760.5

O-36:5 (l) O-36:4 (l) 16:0/ 20:5 (l) 16:0/ 20:4 (h) 18:1/ 18:2 (m) 18:0/ 18:2 (m) 18:0/ 18:1 (m) O-38:5 (l) O-38:4 (l) 18:1/ 20:6 (l)

766.5 768.5 780.5 782.5 784.5 786.5 788.5 794.4 796.5 804.6

16:0/ 22:6 (m) 18:1/ 20:4 (m) 18:0/ 20:4 (h) 18:2/ 22:6 (l)

806.5 808.5 810.5 830.5

18:1/ 22:6 (l) 18:0/ 22:6 (l) 18:0/ 22:5 (l) 18:0/ 22:4 (l) d16:1/ 16:0 (l) d17:1/ 16:0 (l)

832.5 834.5 836.5 838.5 675.4 689.4

d18:1/ 16:1 (l) d18:1/ 16:0 (h)

701.5 703.5

d17:1/ 18:0 (l) d18:1/ 18:3 (l) d18:1/ 18:2 (l) d18:1/ 18:1 (l) d18:1/ 18:0 (l) d17:1/ 20:2 (l) d18:1/ 20:1 (l) d18:1/ 20:0 (l)

717.5 725.6 727.6 729.5 731.6 741.6 757.5 759.5

Class Lyso-PC 12 species

PE 15 species

Lyso-PE 12 species

PS 8 species

Species 16:0/0:0 (h) 18:3/0:0 (l) 0:0/18:3 (l) 18:2/0:0 (l) 18:1/0:0 (l) 18:0/0:0 (m) 0:0/20:5 (l) 0:0/20:4 (l) 0:0/20.3 (l) 0:0/22:6 (l) 22:6/0:0 (l) 0:0/22:5 (l) 16:0/ 18:2 (l) 16:0/ 18:1 (l) 16:0/ 20:4 (m) 18:1/ 18:2 (l) 18:0/ 18:2 (m) 18:0/ 18:1 (l) 16:1/ 22:6 (l) 16:0/ 22:6 (l) 16:0/ 22:5 (l) 18:1/ 20:4 (m) 18:0/ 20:4 (h) 18:2/ 22:6 (l) 18:1/ 22:6 (l) 18:0/ 22:6 (l) 18:1/ 22:5 (l) 16:0/0:0 (l) 18:2/0:0 (l) 18:1/0:0 (m) 18:0/0:0 (h) 0:0/20:5 (l) 0:0/20:4 (h) 20:4/0:0 (l) 0:0/22:7 (l) 0:0/22:6 (l) 22:6/0:0 (l) 0:0/22:5 (l) 0:0/22:4 (l) 18:0/ 18:2 (l) 18:0/ 18:1 (m) 18:1/ 20:4 (l) 18:0/ 20:4 (h) 18:0/ 20:2 (l) 18:0/ 22:6 (l)


m/z ratio 496.3 518.4 520.4 522.4 524.4 542.3 544.3 546.3 568.3 570.3 716.3 718.3 740.4 742.4 744.3 746.3 762.3 764.3 766.3 766.3 768.3 788.3 790.3 792.3 792.3 454.3 478.3 480.3 482.3 500.3 502.3 524.3 526.3 528.3 530.3 788.3 790.3 810.6 812.6 816.3 836.3 23


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d17:1/ 22:0 (l) d18:1/ 22:1 (l) d18:1/22:0 (m) d17:1/ 24:1 (l) d17:1/ 24:0 (l) d18:1/ 24:2 (m) d18:1/ 24:1 (m) d18:1/ 24:0 (m)

773.5 785.5 787.5 799.5 801.6 811.5 813.5 815.5

PI 6 species

18:0/ 22:5 (l) 18:0/ 22:4 (l) 16:0/ 20:4 18:0/ 18:2 18:1/ 18:1 18:0/ 18:1 18:1/ 20:4 18:0/ 20:4

838.3 840.3 857.3 861.3 861.3 863.3 883.3 885.3

Table 1 (continued). Class pPE/oPE 31 species

Species P-16:0/ 18:2 (l) P-18:1/ 16:1 (l) P-16:0/ 18:1 (l) P-18:0/ 16:1 (l) O-16:0/ 18:1 (l) P-16:0/ 18:0 (l) P-16:1/ 20:5 (l) P-16:0/ 20:5 (l) P-16:0/ 20:4(m) P-18:1/ 18:2 (l) P-16:0/ 20:2 (l) P-18:0/ 18:2 (l) P-18:1/ 18:1 (l) P-18:0/ 18:1 (l) P-18:1/ 20:5 (l) P-18:2/ 20:4 (l) P-16:0/ 22:6 (l) P-16:0/ 22:5 (l) P-18:1/ 20:4(m) P-16:0/ 22:4 (l) P-18:0/ 20:4 (h) P-18:1/ 20:3 (l) P-16:0/ 22:3 (l) P-18:1/ 22:6 (l) P-20:3/ 20:4 (l) P-18:0/ 22:6 (l) P-20:1/ 20:5 (l) P-18:0/ 22:5 (l) P-20:1/ 20:4 (l) P-18:0/ 22:4 (l) P-20:0/ 20:4 (l)

m/z ratio 700.5 700.5 702.5 702.5 704.5 704.5 720.6 722.6 724.6 726.6 728.6 728.6 728.6 730.6 748.6 748.6 748.6 750.6 750.6 752.6 752.6 752.6 754.6 774.6 774.6 776.6 776.6 778.6 778.6 780.6 780.6

a

The relative abundance of the peak of each PL in the ESI-MS spectrum related to its class is indicated: h (high, > 80%); m (medium, 20-80%); l (low, < 20%).


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Table 2. Overview of side chain-related fragmentations observed in the ESI-3D-Ion TrapMS/MS spectra of selected standard PLs and used for the assignment of the regiochemistry of PLs detected in MPs and platelets samples. M represents the zwitterionic form in the case of PLs having a phosphocholine head (PC, SM, lyso-PC) and the neutral species in all the other cases. See text and Supporting Information for a discussion of fragmentations. PL class

Precursor ion

Regiochemically diagnostic fragmentation(s) or product ions

PC

[M-CH3]-

SM

[M-CH3]-

Lyso-PC

[M+H]+

Loss of acyl chains as ketenes: prevalence from the sn2 position Loss as a ketene of the acyl chain linked to the NH2 group of sphingosine/sphinganine Loss of water; generation of the phosphocholine ion (m/z 184): [M+H-H2O]+/[phosphocholine]+ > 1.3 for PC(n:m/0:0) species < 0.5 for PC(0:0/n:m) species

PE

[M-H]-

Loss of acyl chains as ketenes: prevalence from the sn2 position

Lyso-PE

[M+H]+

Loss of water; loss of phosphoethanolamine: [M+H-H2O]+/[M+H-phosphoethanolamine]+ = 1.3-1.4 for PE(n:m/0:0) species < 0.5 for PE(0:0/n:m) species

oPE/pPE

[M+H]+

Product ion related to the alkyl/alkenyl chain

Product ion related to the acyl chain

PS

[M-H-serine](MS3)

Loss of acyl chains as fatty acids: prevalence from the sn2 position

PI

[M-H]-

Loss of acyl chains as fatty acids: prevalence from the sn2 position


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Figure 1


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Figure 2


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Figure 3 ACS Paragon Plus Environment

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For TOC only


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Phospholipidomics of Human Blood Microparticles - Analytical

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