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Article
Microarray and mass spectrometry-based Methodology
for lipid profiling of tissues and cell cultures
Roberto Antonio Fernández, Jone Garate, Tarson Tolentino-Cortez, Ainara
Herraiz, Laura Lombardero, Fabien Ducrocq, Rafael Rodriguez-Puertas, Pierre
Trifilieff, Egoitz Astigarraga, Gabriel Barreda-Gómez, and Jose A. Fernandez
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b04529 • Publication Date (Web): 21 Nov 2019
Downloaded from pubs.acs.org on November 22, 2019

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Page 1 of 9 Analytical Chemistry

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8 Microarray and mass spectrometry-based Methodology for lipid
9
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profiling of tissues and cell cultures.
11 Roberto Fernández,1,2 Jone Garate,1 Tarson Tolentino-Cortez,2 Ainara Herraiz,2 Laura Lombardero,3
12
13
Fabien Ducrocq,4 Rafael Rodríguez-Puertas,3 Pierre Trifilieff,4 Egoitz Astigarraga,2 Gabriel Barreda-
14 Gómez2* and José A. Fernández1*.
15 1Department of Physical Chemistry, Faculty of Science and Technology; 3Department of Pharmacology, Faculty of Medicine
16 and Dentistry, University of the Basque Country (UPV/EHU), Barrio Sarriena S/N, Leioa 48940, Spain; 2Research
17 department, IMG Pharma Biotech S.L., BIC Bizkaia (612), 48160-Derio, Spain; 4Univ. Bordeaux, INRA, Bordeaux INP,
18 NutriNeuro, UMR 1286, F-33000, Bordeaux, France.
19
20 ABSTRACT: The recent developments in mass spectrometry have revealed the importance of lipids as biomarkers in the context
21 of different diseases and as indicators of the cell’s homeostasis. However, further advances are required to unveil the complex
relationships between lipid classes and lipid species with proteins. Here we present a new methodology that combines microarrays
22
with mass spectrometry to obtain the lipid fingerprint of samples of different nature in a standardized and fast way, with minimal
23 sample consumption. As a proof of concept, we use the methodology to obtain the lipid fingerprint of 20 rat tissues and to create a
24 lipid library for tissue classification. Then, we combine those results with immunohistochemistry and enzymatic assays to unveil
25 the relationship between some lipid species and two enzymes. Finally, we demonstrate the performance of the methodology to
26 explore changes in lipid composition of the nucleus accumbens from mice subjected to two lipid diets.
27
28
29
30 INTRODUCTION them are related to single cell lipid profiling.14, 22-25 There is an
Mass spectrometry is an analytical technique that is increasing interest in the analysis of lipids, due to the central
31
especially well suited for application in the field of “omics” role that these molecules play in fundamental cellular
32 processes. Furthermore, any alteration in the metabolic stage
33 due to its intrinsic characteristics of high speed,
reproducibility, sensitivity and high dynamic range.1 Certainly, of a cell has a measurable and immediate impact in lipids
34 expression, and therefore, lipids may be good candidates for
the world of omics (genomics, proteomics, metabolomics…
35 etc), requires analytical techniques able to handle hundreds or early biomarkers of diseases.26-30 However, the diverse nature
36 even thousands of samples in an automated (or at least, semi- of lipids makes their study a difficult task. The common lipid
37 automated) way, so that minimal user intervention is sample is an extract from a tissue or a cell culture and contains
38 required.2-12 In this context, efforts have been put in combining polar and non-polar species in very different abundances. The
39 matrix-assisted laser desorption/ionization (MALDI) mass most popular protocols for the analysis of lipid extracts
40 spectrometry (MS) with other technical developments to speed involve the use of HPLC-MS, due to the extra dimension that
up the analysis process by simplifying sample preparation, and the separation stage adds, which mitigates in part the
41
to reduce the total amount of sample required in each analysis. undesired suppression effects caused in the low-abundant
42 species.31 The main drawback of this approach is the relatively
43 For example, an extensive literature exists on implementing
the “lab-on-a-chip” concept in MALDI-MS, especially with large amount of sample required for HPLC analysis. In
44 addition, scanning a single sample usually takes 15-20 min.
applications in the field of proteomics.13-18 Numerous
45 advances have also appeared, focused on the development of On the other hand, MALDI requires substantially less sample,
46 new hydrophilic, hydrophobic (or a combination of both) 0.5 - 1 L, but deposition is usually highly inhomogeneous,
47 surface treatments to concentrate the sample, with the presenting strong changes in concentration between the center
48 subsequent reduction of sample volume and increase in of the sample and the rim.32 This may result in large statistical
49 sensitivity.18 Also promising is the application of fluctuations, unless the whole spot is scanned, increasing
50 desorption/ionization on silicon (DIOS), which does not significantly the analysis time. Certainly, reproducibility is a
51 require matrix for sample analysis or the development of known issue in MALDI mass spectrometry.33
52 microfluidics for on-plate separation and derivatization The methodology presented here includes sample
53 previous to the MALDI-MS analysis.19-21 deposition, scanning and data analysis in order to characterize
54 Despite the important developments achieved for the the lipid expression of tissues and cells (Figure 1). The
analysis of proteins, to the best of our knowledge, the protocol enables fabrication of a collection of arrays
55
advances produced so far in the field of metabolomics, and containing different types of samples, spotted with a
56 commercial piezoelectric microarrayer for liquid handling
more precisely for lipid analysis, are more limited and most of
57 from 60 picolitres. Matrix is then deposited using a
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1 sublimator, creating a uniform layer that ensures a constant tissues under study were quickly removed at 4°C to avoid
2 sample/matrix ratio along the whole array. The protocol gives protein denaturation. Tissue samples were stored at -80 °C.
3 high inter-experiment reproducibility, even for experiments Mice were housed in groups of 4-10 animals in standard
4 carried out in different days, or even different months, with polypropylene cages and maintained in a temperature and
5 consumption of a minimal amount of sample. In this regard, humidity-controlled facility under a 12:12 light-dark cycle
6 even in cases were very limited amounts of sample are (8:00 on) with ad libitum access to water and food. All animal
7 available, it is possible to build several replicate arrays, care and experimental procedures were in accordance with the
8 opening the door to the possibility of combining the results INRA Quality Reference System and to french legislations
9 from lipid analysis with others on the distribution and function (Directive 87/148, Ministère de l’Agriculture et de la Pêche)
of membrane proteins. These data may be used to determine and European (Directive 86/609/EEC). They followed ethical
10
novel relationships between lipids and proteins such as those protocols approved by the Region Aquitaine Veterinary
11 found in the lipid rafts. 34 Services (Direction Départementale de la Protection des
12
As a proof of concept, we present here the results obtained Animaux, approval ID: B33-063-920) and by the animal ethic
13 with microarrays consisting of whole cells, membranes committee of Bordeaux CEEA50. Every effort was made to
14 isolated from a collection of rat and monkey tissues and minimize suffering and reduce the number of animals used.
15 membranes isolated from accumbens nuclei of mice subjected For dietary lipid manipulations, female C57BL6/J mice were
16 to different dietary lipid intake. fed with isocaloric diets containing 5% fat with a high (n-3
17 PUFA deficient diet) or low Linoleic Acid/Alpha-Linolenic
18 Acid ratio (Control diet) across gestation and lactation and
19 offspring were maintained under the same diet after weaning
20 until adulthood.35, 36 For tissue collection, mice were dislocated
21 and the brains quickly removed and snap-frozen in isopentane
22 (Merck) on dry ice and stored at -80°C. Nucleus accumbens
samples were punched (No.18035-01, Fine Science Tools)
23
from 200 µm frozen slices in a cryostat. Tissues were then
24 kept at -80°C until processing.
25
26
27 Cell lines and culture conditions
28 Human primary pancreatic adenocarcinoma (BX-PC3),
29 human colorectal carcinoma (HCT 166) and human
30 fibrosarcoma (HT1080) were purchased from Leitat
Technological Center). Manufacturer laboratories ensure cell
31
lines authenticity and the absence of Mycoplasma
32 contamination. The cells were grown at 37°C in a humidified
33 incubator with a 5% CO2-95% air-water saturated atmosphere.
34 Cells were harvested by trypsinization and after washing (PBS
35 Figure 1. Walkthrough of a MALDI-MS lipid analysis carried out 1X, pH7.4), they were stored at -80C in cryoprotective buffer.
36 with microarrays. Cell membranes isolated from cell lines or Isolation of cell membranes
37 animal tissues, whole cells or a combination of all three types of
Samples were homogenized using a Teflon-glass grinder
38 samples are deposited in the array using a robot. Then, the array is
covered with matrix using a sublimator. The array is ready for (Heidolph RZR 2020) in 20 volumes of homogenized buffer
39 (1 mM EGTA, 3 mM MgCl2, and 50 mMTris-HCl, pH 7.4)
analysis using a mass spectrometer. In our case, an orbitrap-type
40 supplemented with 250 mM sucrose. The crude homogenate
mass spectrometer was used but any other type of mass
41 spectrometer could, in principle, be used. Data from each spot are was subjected to a 1000x g for 8 min, and the resultant
42 then analyzed in an automated way using in-house developed supernatant was centrifuged again at 18000x g for 15 min
43 software based on advanced algorithms. (4°C, Microfuge 22R centrifuge, Beckman Coulter). The pellet
44 was washed in 20 volumes of homogenized buffer using an
45 Ultra-Turrax (T10 basic, IKA) and re-centrifuged under the
METHODS same conditions. The homogenate aliquots were stored at -80
46
Animals °C until they were used. Protein concentration was measured
47
by the Bradford method and adjusted to the required
48 Male Sprague-Dawley rats (250-300 g) were used to build
concentrations.
49 the arrays and for brain tissue sections. Animal were
maintained under standard conditions (food and water ad Cell membrane microarray development
50
51 libitum; 12h/12h light/dark cycle, light on 7 a.m.) and research Membrane homogenates were resuspended in buffer and
protocols were carried out according to guidelines approved printed (4 nL per spot, 3-5 replicates per sample) onto glass
52
by the Faculty of Medicine of the Basque Country University slides using a non-contact microarrayer (Nano_plotter NP
53 ethical committee based on the internationally accepted 2.1).37 Microarrays were stored at -20°C until they were used.
54 directives (86/609/EEC). After being deeply anesthetized with ChE activity detection
55 a ketamine/xylazine cocktail (60 mg/kg of Ketamine-HCl and
56 5 mg/kg Xylazine-HCli.p.), rats were decapitated and the ChE activity was determined adapting the methodology
57 described by Karnovsky and Roots38 to microarrays. Briefly,
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1 microarrays were dried for 60 min and then incubated at room modification of a Hierarchical Divisive Analysis,20 a reliable
2 temperature with the reaction medium (0.1 M Tris-maleate and robust segmentation of the data was achieved. By
3 buffer, 0.1 M sodium citrate, 30 mM cupric sulfate and 50 Mm definition, the RankCompete algorithm divides the experiment
4 potasic ferrocyanide, pH 6.5) in presence of 2.3 Mm into two segments. However, the data from the array may
5 acetylthiocholine iodine as substrate. The incubation medium contain a variable number of segments. Therefore, we used a
6 was protected from light to avoid the substrate degradation. variation of the Divisive Analysis algorithm (DIANA) to
7 The selective butyrylcholinesterase (BuChE) irreversible create a variable number of walkers. Thus, the final software is
8 inhibitor, Iso-OMPA (0.1 mM) was added to the reaction a segmentation algorithm based on DIANA and using
9 buffer to determine AChE activity. RankCompete as split function. Once the segments were
GAPDH immunodetection obtained, the correlation between them was obtained and the
10
value was used to assign a color to each segment using a color
11 Microarrays were incubated with the blocking buffer (PBS,
scale and 1-correlation between the segments. In this way, the
12 0.3% triton and 5% skimmed dried milk) for 30 min before
two segments that present the lowest correlation occupy the
13 adding the anti-GAPDH mouse monoclonal antibody (Merck
two extremes of the scale and those segments with more
14 Millipore) at 0.05mg/ml dissolved in blocking buffer. After 1
similar average spectra receive colors that are closer in the
hour at room temperature, microarrays were reveled with the
15 scale.
secondary antibody goat anti-mouse Alexa Fluor 555 (Abcam)
16 at 1:200 in blocking buffer. Finally, microarrays were rinsed The two unsupervised models were a hierarchical-divisive
17 with PBS for 10 min and the fluorescence was quantified clustering (splitting method: RankCompete; pair-wise
18 using a microarray scanner (Agilent G2565CA). Non-specific distance: the correlation, HDC-RC) and a principal
19 immunoreactivity was determined in the absence of the components analysis (PCA). The supervised models were a
20 primary antibody. Logistic Regression, a Random Forest of 20 trees and a Neural
21 Network with 500 hidden neurons and ReLu as activation
MALDI-MS
22 function. To perform the analysis of the average spectra of the
The arrays were covered with a suitable matrix with the aid spots, all matrix peaks and isotopic distribution peaks were
23 of a standard glass sublimator (Ace Glass 8233), producing a filtered and the resulting lists of peaks were normalized using
24 uniform film of aprox. 0.2 mg/cm2.39 As sublimation is a dry the total ion current (TIC) of the remaining peaks.
25 method, it prevents lipid delocalization, which is important in
Regarding data analysis including positive- and negative-ion
26 this device, taking into account the proximity of the spots in
mode data, MS+ and MS- were normalized separately and
27 the array. 2-mercaptobenzotiazol (MBT) was used for
positive-ion mode and 1,5-diaminonaphtalene (DAN) for then analyze them together. In that way, for each spot, we
28 have the normalized MS- and MS+ mass channel values in the
29 negative-ion mode.40,41 Once introduced in the spectrometer,
the arrays were scanned as in a MALDI-imaging experiment. same row. In the first example, matrix peaks and isotopic
30 distributions were removed, and the remaining peaks re-
The area of the array was explored following a grid of
31 normalized against their total ion current for the PCA.
coordinates separated 150 m. As the spot of the arrays had a
32 However, HDC-RC was done with all the peaks of the
diameter of 450 m, this means that nine pixels were recorded
33 at each spot. The mass spectrometer used in this work was an spectrum higher than the 0.5% of the maximum, in order to
34 LTQ-Orbitrap XL (Thermo Scientific, San José, CA, USA), demonstrate the reproducibility of the arrays. In the second
35 equipped with a MALDI source with a N2 laser (60 Hz, 100 example, all the peak were used for HDC-RC again, but to
36 J/pulse maximum power output). The laser spot is an simplify the t-test, only those peaks with a clear assignment
37 ellipsoid of aprox. 50-60 m x 140-160 m.39 Thus, the size of were considered and the spectra were re-normalize against
38 the spots in the array were chosen as to avoid laser shot the total number of lipid ions. The rest were removed,
39 overlapping. Two microscans of ten shots were used to including matrix clusters, fragments and isotopic
40 produce the spectrum of each pixel. Mass resolution was set to distributions.
41 30000 at m/z = 400 in an 550-950 observation window, both
42 in positive- and negative-ion modes.
43 The spectra were analyzed as in a MALDI-imaging
44 experiment: the whole set of data were loaded as a single
45 experiment. Therefore, data loading included spectra
normalization by total ion current (TIC), spectra alignment
46
and peak picking, filtering all the m/z with intensity < 0.5 % of
47
the strongest peak in the spectrum. Statistical analysis was
48 carried out using an in-house made statistical algorithm, based
49 on rank compete (see below) and built in Matlab (MathWorks,
50 Natick, USA), for segmentation, and Orange Biolab42 for
51 supervised analysis.
52 Statistical Analysis
53 Segmentation of the pixels in the image of the array was
54 done using a segmentation algorithm, RankCompete,43 based
55 on the properties of Markov Chains44 to define Random
56 Walkers45 competing to divide the imaging experiment into
57 two segments. Using this algorithm as splitting algorithm in a
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1 Figure 2. Lipid fingerprint of the lipid membranes of 20 rat individual pixels, and not the average over the whole spot. As
2 tissues using microarrays and mass spectrometry. a) Bradford each spot presents an individual color, this means that all nine
3 staining of the array. Three replicates of each sample were pixels that compose each spot present very similar spectra.
4 printed, amounting for 78 spots; b) tissues included in the array; Furthermore, all the spots containing replicates of the same
5 c) relative abundance in the samples of some example lipids, sample also correlate, pointing once more to a good intra- and
using a gray scale; d) mass spectra from five example tissues; e) inter-experiment reproducibility.
6
segmentation analysis of the results from five arrays using a rank
7 compete algorithm (see on line methods and supplemental Regarding the lipid signatures of the tissues, all samples
8 material Figures S1-S3). Colors were assigned using the color from central nervous system (CNS) presented spectra with a
9 scale also shown and using the correlation between the spectra: higher degree of correlation, while peripheral tissues (PT) had
10 those pixels with colors closer in the scale present spectra that are distinctive lipid fingerprints, as demonstrated by the different
11 more similar. Cx: brain cortex; HPCP: hippocampus; CBLO: colors assigned by the algorithm and the scores plot of the
Cerebellum; HPTLM: Hypothalamus; CPU: Caudate putamen; three first components of a PCA analysis (see supplemental
12
Spinal Cord Cerv: spinal cord cervix; Printing sol.: printing Figure S1).
13 solution (blank). Cortex concentrations correspond to serial
14 An improved unsupervised classification of the tissues was
dilutions from the original sample (10mg/mL), resulting in obtained when those from CNS were analyzed separately from
15 different total amounts of lipid membranes per spot. The sample is
PT (see supplemental Figures S2 and S3). In this way, a better
16 a pool from 10 different animals.
sensibility towards changes in lipid profile was achieved,
17 RESULTS demonstrating that each tissue has a distinctive and
18 representative lipid fingerprint.
Lipid membranes from different rat tissues
19 The supervised models built with the data from six arrays
20 To demonstrate the capabilities of the methodology, we
created an array containing three replicates of lipid membranes were validated with a 10-fold cross validation, obtaining a
21 classification accuracy of 1, 0.981 and 0.991 for Logistic
from 20 different rat tissues (see Figure 2 and on line materials
22 and methods). The array also included lipid membranes from Regression, Random Forest and Neural Network respectively.
23 rat cerebral cortex at four different concentrations to evaluate To further test the specificity and classification ability of the
24 the quantitative character of the results. A last line of cortex model, we designed four new arrays containing “problem”
25 was included in the lower-right corner of the array, to detect tissues from male rats, female rats, mixtures of tissues, several
26 possible changes in the signal intensity during the experiment new tissues not included in the original array and monkey
27 due to fluctuations in the laser intensity, matrix sublimation or tissues. The arrays were scanned following the above-
any other similar methodological problem. A line containing described methodology and the spectrum of each tissue was
28
printing solution was also included as blank for comparison. classified using the model built with the array in Figure 2. The
29 detailed results may be found in Table S1. If the model is
30 The array was read with the mass spectrometer as in a
MALDI-imaging experiment, which means that a grid of valid, male rat tissues should be correctly classified, as the
31 array used to build the model contained exclusively tissues
coordinates was defined over the area of the array and the
32 mass spectrometer recorded one spectrum at each position. from male rats. Close examination of the results in the table
33 Integration of the mass channels in the spectra and shows that male rat tissues were correctly classified by the
34 representation of the integral against the coordinates using a logistic regression algorithm and the neural network, while the
35 gray scale resulted in the images in Figure 2c. The distance random forest performance was far from being acceptable.
36 between points in the grid was 150 m, resulting in pixels of Both, logistic regression and random forest had problems
37 the same size in the image. As the printing spot size was 450 x classifying female tissues, while neural network showed a
higher success rate. This result may point to the existence of
38 450 m, this means that each spot is represented by nine
noticeable differences between the lipidome of male and
39 pixels. The robot used for sample deposition delivers 300
female rats, especially in tissues from the CNS.
40 pL/drop and 10 drops were deposited at each spot, amounting
for 30nL at each spot. Despite the small amount of sample Finally, the model was tested against mixtures of tissues,
41
deposited, it was enough to record reproducible spectra with a tissues that were not present in the original arrays and tissues
42 from monkey (Figure S4). In most cases, the algorithms
good s/n ratio, as highlighted in Figure 2d.
43 classified the samples into several categories, including some
44 To test the intra and inter experiment reproducibility two
of the components of the mixtures. Interestingly, the neural
45 unsupervised and three supervised models were used (see on
network classified monkey lung as (rat) lung, pointing to
line methods). The results from the analysis of six arrays
46 smaller differences in the lipidome of this organ between rat
scanned along two weeks may be found in Figures 2e and S1-
47 S3. Figure 2 e shows the segmentation of five experiments at
and monkey than in the case of brain or heart (Table S1).
48 once using the divisive hierarchical clustering with
49 RankCompete (HDC-RC) segmentation algorithm directly
50 over the TIC-normalized MS data. The algorithm colors the
51 pixels following the scale shown in the figure using 1 minus
52 the correlation between pixels. In this way, pixels with the
53 same color present similar spectra and those pixels with colors
54 that are very distant in the scale contain very different spectra.
55 Actually, the colors at the two ends of the scale are assigned to
the two pixels with the lowest correlation. One must take into
56
account that the analysis was done using the spectra of the
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1 cluster classification) and 15 animals fed with the control diet

2 (light blue spots in the 2 cluster and red spots in the 300
3 cluster classification). An additional group of four spots
4 containing only printing solution (p.s.) was also included as
5 blank. The HDC-RC algorithm was used for the segmentation;
6 b) Relative abundance of four lipid species that show
7 significant changes between the two groups of animals; c) PE
8 species that experience the largest changes in the spectra
9 recorded in negative-ion mode between both diets. Other
significant species can be found in supplementary material
10
(Table S2).
11
Analysis of the expression of the rest of the sulfatides
12
detected in the array showed that SFT d36:1 and t40:1 are
13 always in less abundance than d42:2, d42:1 and t42:1 in CNS.
14 Such ratio is not preserved in other tissues, though. Those
15 ratios may be pointing to conserved metabolic pathways
16 among different cells or to the presence of a specific type of
17 cell, which would be the responsible for the maintenance of
18 Figure 3. Using arrays to uncover patterns in the lipid SFT levels.
19 expression. a) PC 32;0 and sulfatide (SFT) D42:2 present The results from those analyses may be complemented with
20 completely opposed distributions in rat brain tissue sections; further results from proteomic assays, performed using
21 b) The same negative correlation was found in the arrays of consecutive arrays. For example, comparison between
lipid membranes; c) The controlled deposition of membrane expression of SFT d36:1 and that of glyceraldehyde-3-
22
homogenates in the array allowed us to quantify the phosphate dehydrogenase (GAPDH) determined by
23 correlation between the two lipids in CNS tissues; d)
24 immunochemistry showed a high degree of correlation (R =
Additionally, a conserved ratio between expression of five 0.85, Figure S5). Likewise, a correlation between the same
25 SFT species was found. Images in tissue recorded at 50 lipid species and the activity of the cholesterase (ChE)
26 m/pixel in negative-ion mode. determined by enzymatic assays in consecutive cell membrane
27 Exploring lipid expression patterns along tissues microarrays unveiled also a reasonable correlation (R = 0.79,
28 Figure S6) in tissues from CNS.
An additional advantage of having the lipid profile of
29 several tissues in a single experiment and in standardized Diet influence on brain lipid composition in mice
30 conditions is that it enables comparative studies that may As a final test, we used the array technology in a study to
31 unveil patterns in lipid expression across multiple tissues. For determine the influence of dietary manipulations on the lipid
32 example, Figure 3a shows the distribution of two composition of mouse brain. The study consisted of two
33 representative lipids, PC 32:0 and SFT d42:2, along a coronal groups of animals: one fed a n-3 polyunsaturated fatty acids
34 section of rat brain. These two lipids have a complementary (PUFA)-deficient diet (sunflower oil; n-3 deficient) while the
35 distribution in all brain areas in the section. The same negative control group was fed a PUFA-balanced diet (rapeseed oil;
36 correlation was found in the array, with the difference that the control). It is well known that various psychiatric disorders,
37 tissue section experiment took over 14 hours, while the such as schizophrenia, obsessive-compulsive disorder or
experiment in the array only required of 2 hours of measuring attention deficit hyperactivity disorder, are associated with a
38
time. Furthermore, as the data in the array are normalized to dysfunction of the reward system and lipid composition.46-50
39 the total amount of protein, it is possible to quantify the
40 To check the influence of dietary manipulations on lipid
correlation between those two lipids (Figure 3c), highlighting composition of a specific brain area involved in reward
41 that their abundance maintains and excellent correlation processing, the nucleus accumbens of 15 animals fed with the
42 among the tissues of the CNS. control diet and of 16 n-3 PUFA deficient animals were
43 dissected and their lipid membranes used to build an array
44 containing four replicates from the nucleus accumbens of each
45 animal (Fig. 4). Despite the small size of that anatomic area
46 (1-3 mg), the amount of lipid membranes extracted was
47 enough to build 44 arrays. Three of those arrays were scanned
48 in positive-ion mode and another three in negative-ion mode.
49 The lipid signature of the lipid membranes of the nucleus
50 accumbens was highly reproducible and specific of each group
51 of animals, as highlighted by the statistical analysis shown in
52 Figure 4a, where all the spots from the control group appeared
53 Figure 4. Lipidomic analysis of the nucleus accumbens of in the same cluster (white), while those from the n-3 PUFA
mice fed with different diets. a) Classification of the lipid deficient group appeared in a blue-colored cluster (2 cluster
54
fingerprints in a cell membrane microarray consisted of four classification). The spectra of the printing solution (p.s.,
55 lower-right corner) did not show in any of the two clusters, as
replicate of 16 animals fed with n-3 PUFA deficient diet
56 a further confirmation of the analysis. The large number of
(white spots in the 2 cluster and dark blue spots in the 300
57 replicates allowed us to identify, with a high degree of
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1 confidence, the species that marked the difference between the
2 two groups. Certainly, the data in Figure 4b and 4c show a
3 clear increase in species containing 5 unsaturation in the
4 spectra of the n-3 PUFA deficient group, while species
5 containing 6 unsaturation were more abundant in the control
6 group.
7 DISCUSSION
8 of GAPDH/SFT d36:1 and ChE/SFT d36:1 along the tissues
The present study describes a new methodology to record
9 suggest not only a co-localization but also a common function.
the lipid profile of cells and membrane extracts for lipid
10 biomarker discovery, also enabling combined studies of lipid
11 composition and protein function. The methodology uses the
12 advantages of microarray technology, mass spectrometry and
13 bioinformatics in an analytic platform. The results presented
14 demonstrate its strength in lipid fingerprint determination,
tissue classification, and identification of lipid changes
15
associated to dietary manipulation. In the first example
16 presented, the connection between each type of tissue and its
17 lipid signature was clearly demonstrated, highlighting that
18 each tissue has a characteristic lipid fingerprint, even when
19 only those lipids in the membranes are used instead of the
20 whole lipid content of the cell. Different tissues present
21 different membrane lipidome, but also the same tissue from
22 different animals (rat vs monkey) or even from animals of
different gender (male/female rats) exhibit differences in the
23
membrane lipidome.
24
Using lipid membranes instead of lipid extracts has two
25
advantages: the sample preparation protocol is simpler and the
26
27 membrane proteins remain fully functional, and therefore the
results from lipid analysis may be combined, as demonstrated
28
in the present work, with other experiments, such as enzymatic
29 assays or immunohistochemistry, creating a powerful tool to
30 unveil complex relationships between lipid composition and
31 protein activity. Such connections have been sparingly
32 explored, due to the inherent difficulties of the studies.
33 Another important aspect is that the small size of the spots
34 permits a fast reading of the data. When standard MALDI
35 targets are used for lipid analysis, strong variations in
36 composition between the center and the edge of the spot are
37 usually found,32 resulting in spectra that are not representative
38 of the real composition of the sample, unless the whole well is
39 thoroughly scanned. This may require hundreds of laser shots,
40 while the spots in the arrays require measuring only nine
pixels, taking 20 laser shots/pixel, significantly reducing the
41
acquisition time, an important factor, especially when hi-
42 resolution/low speed mass analyzers are used. The small size
43 of the array also allowed us to cover the whole surface with
44 matrix using a sublimator, which ensures a very uniform
45 matrix deposition, resulting in more uniform signal intensity.
46 Moreover, this methodology substantially reduces not only the
47 intra-, but also the inter-experimental variability, enabling the
48 combined analysis of experiments recorded in different days.
49 In the first proof of concept, we determined a lipid fingerprint
for 20 different tissues, creating a database that enabled tissue
50
classification with accuracies over 0.98. Only six microarrays
51
were required for this experiment, resulting in 12 hours of
52 total scanning time with our mass spectrometer. Moreover, the
53
bioinformatic analysis revealed groups of lipids (clusters) that
54 presented particularly strong correlations among the analyzed
55 tissues. This observation suggests the existence of conserved
56 lipid metabolism in those tissues, or the presence of a specific
57 type of cell. For example, the cluster of the sulfatides could
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Page 6 of 9
indicate the proportion of white matter cells present in the
different areas of the CNS.51, 52 If such is the case, these lipids
could be used as specific biomarkers of this kind of cells,
improving the sensibility obtained with the lipid fingerprint as
in evolved DNA tests. However, some of these correlations
could also refer to structural or metabolic relationships such as
those present in lipid rafts.34 In this sense, the constant ratios
According to this point, a reduction in the expression and
density of these proteins and sulfatides has been described by
independent studies in neurodegenerative diseases such as
Alzheimer disease.53-55 These evidences indicate a relationship
between these specific membrane components, although
further experiments are required to confirm it.
Regarding the exploration of changes in lipid profile related
to dietary manipulation, the advantage of a low per spot
sample deposition without a significant reduction in the S/N
ratio and in the reproducibility was clear, as it turned small
changes in low intensity signals into statistically significant
variations. As already stated, the arrays were prepared and
stored for several weeks under nitrogen atmosphere at -80 ºC
before scanning using MALDI-MS and data recording was
carried out along several days. Still, data were consistent and
reproducible, pointing to a high stability of the arrays.
Last but not least, as mentioned in the introduction, the
content of the arrays may not be limited to lipid membranes
but they may include lipid homogenates or whole cells,
enabling fast characterization of the identity of cell cultures or
the changes in their lipidome in response to different
alterations (see Figure S7).
CONCLUSIONS
We present here an improved methodology to analyze the
lipidome of biological samples. In summary, it is a
miniaturized version of the traditional MALDI-MS protocol
that results in smaller sample consumption per analysis, a
higher reproducibility and fast data analysis thanks to an
improved algorithm for statistical analysis. In addition, it
highlights the specificity of lipid composition in tissues and
cells and opens the possibility of combining MS results with
other techniques in consecutive experiments.
ASSOCIATED CONTENT
Supporting Information
Additional figures of tissue classification and correlation between
lipid species; tables with tissue classification by comparison with
the lipid fingerprint of the tissues in the rat array; table with the
species that experience the largest changes between both mice
models.
The Supporting Information is available free of charge on the
ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
* Dr. Gabriel Barreda-Gómez, Research department, IMG Pharma
Biotech S.L., BIC Bizkaia (612), 48160-Derio, Spain. Phone:
+34944316577. email: Gabriel.barreda@imgpharma.com
6
Page 7 of 9 Analytical Chemistry

1 * Dr. José A. Fernández, Department of Physical 13 Urban, P. L.; Amantonico, A.; Zenobi, R. Lab-on-a-plate:
2 Chemistry, Faculty of Science and Technology, University Extending the functionality of MALDI-MS and LDI-MS targets.
3 of the Basque Country (UPV/EHU), Barrio Sarriena S/N, Mass Spectrom. Rev. 2011, 30, 435-478.
4 14 Mao, S.; Li, W.; Zhang, Q.; Zhang, W.; Huang, Q.; Lin, J.
Leioa 48940, Spain. Phone: +34946015387; Fax: Cell analysis on chip-mass spectrometry. TrAC Trends in Analytical
5 +34946013500. Email: josea.fernandez@ehu.es Chemistry. 2018, 107, 43-59.
6 15 Lee, J.; Soper, S. A.; Murray, K. K. Microfluidics with
7 Present Addresses MALDI analysis for proteomics—A review. Analytica Chimica Acta.
8 2009, 649, 180-190.
†If an author’s address is different than the one given in the
16 He, X.; Chen, Q.; Zhang, Y.; Lin, J. Recent advances in
9 affiliation line, this information may be included here.
microchip-mass spectrometry for biological analysis. TrAC Trends in
10 Analytical Chemistry. 2014, 53, 84-97.
Author Contributions
11 17 Pedde, R. D.; Li, H.; Borchers, C. H.; Akbari, M.
The manuscript was written through contributions of all authors.
12 All authors have given approval to the final version of the
Microfluidic-Mass Spectrometry Interfaces for Translational
13 Proteomics. Trends in Biotechnology. 2017, 35, 954-970.
manuscript. 18 Oedit, A.; Vulto, P.; Ramautar, R.; Lindenburg, P. W.;
14 Hankemeier, T. Lab-on-a-Chip hyphenation with mass spectrometry:
15 ACKNOWLEDGMENT strategies for bioanalytical applications. Current Opinion in
16 This research was supported by Spanish Ministery of Economy Biotechnology. 2015, 31, 79-85.
17 and Competition (MINECO/FEDER, grant n. RTC-2015-3693-1) 19 Lowe, R. D.; Szili, E. J.; Kirkbride, P.; Thissen, H.; Siuzdak,
and the Basque Government (IT1162-19). JG thanks the Basque G.; Voelcker, N. H. Combined Immunocapture and Laser
18 Desorption/Ionization Mass Spectrometry on Porous Silicon. Anal.
19 Government for a pre-doctoral fellowship. PT was supported by
Chem. 2010, 82, 4201-4208.
INRA and University of Bordeaux, Idex Bordeaux “chaire
20 20 Liu, Q.; Guo, Z.; He, L. Mass spectrometry imaging of small
d’installation” (ANR-10-IDEX-03-02), NARSAD Young molecules using desorption/ionization on silicon. Anal. Chem. 2007,
21 Investigator Grants from the Brain and Behavior Foundation, 79, 3535-3541.
22 ANR “SynLip” (ANR-16-CE16-0022) and Region Nouvelle 21 Peterson, D. S. Matrix-free methods for laser
23 Aquitaine 2014-1R30301-00003023. FD held a PhD fellowship desorption/ionization mass spectrometry. Mass Spectrom. Rev. 2007,
24 from the French research ministry. 26, 19-34.
25 22 Chen, Q.; He, Z.; Liu, W.; Lin, X.; Wu, J.; Li, H.; Lin, J.
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