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Analysis of plant nucleotide sugars by hydrophilic interaction liquid chroma-tography and tandem mass spectrometry
Jun Ito, Thomas Herter, Edward E.K. Baidoo, Jeemeng Lao, Miguel E. Vega-Sánchez, A. Michelle Smith-Moritz, Paul D. Adams, Jay D. Keasling, BjörnUsadel, Christopher J. Petzold, Joshua L. Heazlewood
PII: S0003-2697(13)00570-8DOI: http://dx.doi.org/10.1016/j.ab.2013.11.026Reference: YABIO 11577
To appear in: Analytical Biochemistry
Received Date: 23 August 2013Revised Date: 11 November 2013Accepted Date: 22 November 2013
Please cite this article as: J. Ito, T. Herter, E.E.K. Baidoo, J. Lao, M.E. Vega-Sánchez, A. Michelle Smith-Moritz,P.D. Adams, J.D. Keasling, B. Usadel, C.J. Petzold, J.L. Heazlewood, Analysis of plant nucleotide sugars byhydrophilic interaction liquid chromatography and tandem mass spectrometry, Analytical Biochemistry (2013), doi:http://dx.doi.org/10.1016/j.ab.2013.11.026
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1
Analysis of plant nucleotide sugars by hydrophilic interaction liquid chromatography and 1
tandem mass spectrometry 2
3
Jun Itoa, Thomas Hertera,b, Edward E. K. Baidooa, Jeemeng Laoa, Miguel E. Vega-Sáncheza, A. 4
Michelle Smith-Moritza, Paul D. Adamsa,c, Jay D. Keaslinga,c,d, Björn Usadelb,e,f, Christopher J. 5
Petzolda and Joshua L. Heazlewooda,* 6
7 aJoint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National 8
Laboratory, Berkeley, California, 94720, USA 9 bMax Planck Institute of Molecular Plant Physiology, Potsdam-Golm 14476, Germany 10 cDepartment of Bioengineering, University of California, Berkeley, California 94720, USA 11 dDepartment of Chemical and Biomolecular Engineering, University of California, Berkeley, 12
California, 94720, USA 13 eRWTH Aachen University, Institute for Biology I, Aachen 52056, Germany 14 fForschungszentrum Jülich, IBG-2: Plant Sciences, Jülich 52425, Germany 15
16
17
*Corresponding Author 18
Joshua L. Heazlewood 19
Joint BioEnergy Institute 20
Lawrence Berkeley National Laboratory 21
One Cyclotron Road MS978-4466 22
Berkeley, CA 94720, USA. 23
Ph +1 510 495 2694 24
Fax +1 510 486 4253 25
Email: [email protected] 26
27
Running Title: Analysis of plant nucleotide sugars by LC-MS/MS 28
29
Subject Category: Metabolite Determinations 30
2
Abstract 31
Understanding the intricate metabolic processes involved in plant cell wall biosynthesis is 32
limited by difficulties in performing sensitive quantification of many involved compounds. 33
Hydrophilic interaction liquid chromatography is a useful technique for the analysis of 34
hydrophilic metabolites from complex biological extracts and forms the basis of this method to 35
quantify plant cell wall precursors. A zwitterionic silica-based stationary phase has been used to 36
separate hydrophilic nucleotide sugars involved in cell wall biosynthesis from milligram 37
amounts of leaf tissue. A tandem mass spectrometry operating in selected reaction monitoring 38
mode was used to quantify nucleotide sugars. This method was highly repeatable and quantified 39
twelve nucleotide sugars at low femtomole quantities, with linear responses up to four orders of 40
magnitude to several 100 picomoles. The method was also successfully applied to the analysis of 41
purified leaf extracts from two model plant species with variations in their cell wall sugar 42
compositions and indicated significant differences in the levels of six out of twelve nucleotide 43
sugars. The plant nucleotide sugar extraction procedure was demonstrated to have good recovery 44
rates with minimal matrix effects. The approach results in a significant improvement in 45
sensitivity when applied to plant samples over currently employed techniques. 46
47
Keywords 48
nucleotide sugars; plant cell walls; hydrophilic interaction liquid chromatography; Arabidopsis; 49
rice; selected reaction monitoring 50
3
Introductory Statement 51
Plant cell walls have been the focus of recent efforts to convert biomass into liquid transportation 52
fuels with the intention of providing a sustainable alternative to fossil fuels [1]. The majority of 53
nucleotide sugar substrates of plant cell wall polymers are synthesized through a series of 54
nucleotide sugar interconverting enzymes in the cytosol and Golgi apparatus from UDP-α-D-55
glucose or GDP-α-D-mannose [2]. Cell wall UDP-sugar precursors include UDP-α-D-glucose 56
(UDP-Glc), -α-D-galactose (UDP-Gal), -α-D-glucuronate (UDP-GlcA), -α-D-galacturonate 57
(UDP-GalA), -α-D-xylose (UDP-Xyl), -α-D-apiose (UDP-Api), -β-L-arabinose (UDP-Ara) and -58
β-L-rhamnose (UDP-Rha) [3]. Cell wall GDP-sugar precursors include GDP-α-D-glucose (GDP-59
Glc), -α-D-mannose (GDP-Man), -β-L-Galactose (GDP-Gal) and -β-L-fucose (GDP-Fuc) [3]. 60
Other nucleotide sugar precursors include CMP-D-ketodeoxyoctonate (CMP-Kdo), which is an 61
activated acid sugar that is incorporated into pectic rhamnogalacturonan II fractions of primary 62
cell walls of higher plants [4]. There are also numerous minor nucleotide sugars that also exist in 63
plants, although evidence for their incorporation into plant cell walls is rare or unknown [5]. 64
Glycosyltransferases utilize these activated nucleotide sugar donors as substrates to produce the 65
major plant cell wall polysaccharides; cellulose, and matrix polysaccharides (e.g. hemicellulose 66
and pectin) [6]. 67
Reverse genetic studies of enzymes involved in cell wall biosynthesis using the model dicot plant 68
Arabidopsis thaliana have shown that nucleotide sugars involved in cell wall biosynthesis are 69
essential for normal plant growth and development. For example, the mur1 mutant is a non-70
functional cytosolic GDP-D-mannose 4', 6’-dehydratase catalyzing the first step in converting 71
GDP-Man into GDP-Fuc. In the pectic component rhamnogalacturonan-II of Arabidopsis mur1 72
mutants, GDP-Fuc is substituted with GDP-Gal, preventing the formation of borate-dependent 73
4
dimers and mur1 plants display dwarf phenotypes [7]. In addition, the mur4 mutant is a Golgi-74
targeted UDP-xylose 4'-epimerase partially defective in the last step of UDP-Ara synthesis. 75
Analysis of leaf samples from Arabidopsis mur4 plants show a 50% decrease in L-arabinose in 76
its cell wall compared with leaves from wild-type plants [8; 9]. Finally, knock-out mutants of 77
two nucleotide sugar mutases (RGP1 and RGP2) that interconvert UDP-L-arabinopyranose 78
(UDP-Arap) to UDP-L-arabinofuranose (UDP-Araf) have markedly lower total L-arabinose 79
content (12 to 31%) compared with wild-type plants [10]. Down regulation of their expression 80
levels was detrimental to the development of affected plants, along with virtually no L-arabinose 81
in their cell walls [10]. 82
Measuring metabolic changes within the network of cell wall biosynthetic reactions is difficult 83
because of the high number of metabolites involved. Metabolic analysis of plant cell wall 84
biosynthesis requires a highly sensitive and robust method to detect changes in levels of 85
precursors such as nucleotide sugars. Recently a number of quantitative methods based on 86
porous graphitic carbon (PGC) [11] , anion exchange [12; 13] and ion-pair reversed-phase 87
chromatography [14] coupled to mass spectrometry (MS) have been developed. Several of these 88
approaches have been employed to directly measure nucleotide sugars from plant material, 89
namely Arabidopsis thaliana cell cultures and rosette leaves [11; 12]. 90
An emerging chromatographic method has gained interest for its capacity to separate polar 91
metabolites from complex biological mixtures. Hydrophilic Interaction Liquid Chromatography 92
(HILIC) consists of a polar chromatographic surface with the starting mobile phase containing 93
low aqueous content in low-polarity solvent (i.e. acetonitrile) [15; 16]. Polar compounds are 94
retained in the water-rich hydrophilic stationary phase away from the solvent mobile phase. They 95
are eluted in order of increasing polarity in higher aqueous conditions in the mobile phase [16]. 96
5
Low overall aqueous content (typically 5 to 40%) and the limited amount of salts required in the 97
mobile phase solutions allow HILIC to be highly compatible with electrospray ionization (ESI) 98
mass spectrometry. A recent metabolomic study applied zwitterionic silica (ZIC)-based 99
stationary phase in HILIC mode to separate and quantify over 200 hydrophilic intracellular 100
metabolites, including several nucleotide sugars from extracts of β-lactam antibiotic fermentation 101
broths demonstrated the utility of the separation approach [17]. 102
Here, we present a highly sensitive and robust LC-MS/MS method using ZIC-HILIC coupled 103
with a triple quadrupole operating in multiple reaction monitoring mode to compare nucleotide 104
sugar levels from leaves of two plants with different cell wall compositions; the model dicot 105
plant species, Arabidopsis thaliana and the model monocot plant species rice (Oryza sativa). 106
6
Materials and Methods 107
Nucleotide sugar standards and reagents 108
All chemicals were analytical grade or higher and were used as received without any further 109
purification. Nucleotide sugar standards were obtained from the following sources: UDP-α-D-110
xylose (p), UDP-β-L-arabinose (p), UDP-α-D-galacturonic acid (p) (Carbosource Services, 111
Complex Carbohydrate Research Center, Athens, GA); UDP-α-D-glucuronic acid (p), UDP-α-D-112
glucose (p), UDP-α-D-galactose (p), UDP-N-acetyl-α-D-glucosamine, UDP-N- acetyl-α-D-113
galactosamine, GDP-α-D-mannose (p), GDP-β-L-fucose, GDP-α-D-glucose (p) (Sigma-Aldrich, 114
St. Louis, MO); UDP-β-L- arabinose (f) (Peptides International, Louisville, KY). 115
116
Plant growth and sample harvest 117
Arabidopsis thaliana (Col-0) plants were grown with 16 hour photoperiod at 22ºC with 90 μmol 118
m−2 s−1 illumination intensity during the day period. Arabidopsis rosettes from 4-week old plants 119
(three separate individuals) were sampled simultaneously in the middle of the light period. These 120
were immediately frozen in liquid nitrogen and stored at -80 °C until used for metabolic 121
extraction. Rice (Oryza sativa, cultivar Nipponbare) plants were grown in chambers under the 122
following conditions: 12 hour daylight, 470 μmol m−2 s−1 illumination intensity, 80% relative 123
humidity, 26 °C for 1 h at the beginning and end of the cycle, and 28 °C for the remaining 10 h; 124
12 h dark, 80% relative humidity, 26 °C. Leaf material from three individual plants (4-5 weeks 125
old) was sampled in the middle of the day period. These were immediately frozen in liquid 126
nitrogen and stored at -80 °C until used for metabolic extraction. 127
7
128
Monosaccharide composition analysis of extracted cell wall material 129
Leaf material from three individual plants of either rice (4-5 week old) or Arabidopsis (4-week 130
old) was harvested during the day period and dried in an oven at 40ºC for three days. Dried 131
material was ground with a bead beater (Retsch, GmbH, Germany) to a fine powder at 30 Hz for 132
1 to 2 minutes. Preparation and hydrolysis of alcohol-insoluble residues from ground material 133
were separately prepared from three independent biological replicates for both Arabidopsis and 134
rice as previously outlined [18]. Monosaccharide composition was measured by high-135
performance anion exchange chromatography with pulsed amperometric detection (HPAEC-136
PAD, Dionex, Sunnvale CA) using a CarboPac PA20 column as previously outlined [18]. 137
138
Nucleotide sugar extraction from plant material 139
After grinding the frozen leaf material to a fine powder using a bead beater (Retsch GmbH, 140
Germany), nucleotide sugars were extracted from 10 mg fresh weight (FW) as previously 141
described [19]. The freeze-dried extract was dissolved in 1.2 mL of 10 mM ammonium 142
bicarbonate before using ENVI-Carb SPE column (Sigma-Aldrich, St. Louis, MO) using a 143
previously established purification protocol for bacterial samples [20]. Purified extracts were 144
dried in a CentriVap Vacuum Concentrator System (Labconco, Kansas City, MO) and 145
immediately stored at −80°C. 146
147
Hydrophilic interaction liquid chromatography (LC-MS/MS) 148
8
Metabolite extracts were initially reconstituted in 10 µL of 10 mM ammonium acetate (pH 7) 149
and then diluted 1:10 with a solution of 94% acetonitrile and 10 mM ammonium acetate (pH 7) 150
to produce ~85 % acetonitrile. Thus, FW equivalents of 300 to 700 µg of extracts (10 µL) in 151
~85% acetonitrile, 10 mM ammonium acetate (pH 7) were used for analysis by LC-MS/MS. 152
Liquid chromatography was performed on an 1100 series capillary HPLC system (Agilent 153
Technologies, Santa Clara CA) with a 20 µL flow sensor, a 40 µL sample loop and appropriate 154
capillaries for the flow rate used. During chromatographic runs, the injection volume was 10 µL, 155
plate cooler temperature was set to 10 °C and column compartment was 50 °C. Nucleotide 156
sugars were separated with a ZIC-HILIC stationary phase column (150 mm × 1 mm, 3.5 μm, 157
200Å) and a ZIC-HILIC guard column cartridge (5 mm x 1 mm, 5 μm, 200Å) (Merck SeQuant, 158
Umeå Sweden). The flow rate was 20 μL/min with the mix rate set at 400 μL/min and coupled 159
directly to the mass spectrometer for analysis. The mobile phase was 10 mM ammonium acetate 160
(pH 7), in (A) 90% acetonitrile and (B) H2O. At the start of the run, (A) was set at 85% for 2 161
minutes. Gradient elution was performed starting with 85% (A) to 45% (A) over a period of 15 162
min, then back to starting conditions (45 to 85% A) in 15 min, followed by a re-equilibration 163
period (85% A) of 10 min (total run time 42 min). An extended gradient (15 min) was used for 164
re-equilibration after all as recommended by the ZIC-HILIC column supplier (Merck SeQuant, 165
Umeå Sweden). 166
167
Reverse phase ion pair chromatography (LC-MS/MS) 168
Liquid chromatography was performed on an 1100 series HPLC system (Agilent Technologies, 169
Santa Clara CA). The injection volume was 10 µL for three dilutions (1:2, 1:5 and 1:10) of plant 170
9
metabolite extracts and the nucleotide sugars were separated using a reverse-phase Synergi 4u 171
Hydro 80A-RP 150 x 1 mm column (Phenomenex, Torrance, CA) with a Micro-GuardTM 14 x 1 172
mm cartridge (Alltech Associates, Deerfield, IL) at a temperature of 24 °C. Separation of UDP-173
Glc from UDP-Gal was performed with an equilibration step of 10 min, followed by 35 min 174
isocratic flow of 20 mM buffered triethylamine / acetic acid (TEAA) (pH 6) at a flow rate of 50 175
µL/min into the mass spectrometer. 176
177
Electrospray ionization tandem mass spectrometry 178
For detection of nucleotide sugars separated by ZIC-HILIC, a 5500 QTRAP® LC/MS/MS 179
system (AB Sciex, Foster City, CA) equipped with a TurboIonSpray ion source was used. 180
Specific compound-dependent MS parameters for each nucleotide sugar were determined by 181
direct infusion at the MS interface of individual standards dissolved in 50% acetonitrile 182
(concentration of 1 pmol µL-1) at a flow rate of 20 µL min-1. Declustering potential (DP), 183
entrance potential (EP) and collision energy (CE) were adjusted for Q1/Q3 transitions. The 184
specific precursor mass [M-H]-, product ions and collision energies applied are compiled in 185
Table 1. The 5500 QTRAP® system was operated in negative ion mode using the multiple 186
reaction monitoring (MRM) scan type. The ion spray voltage was set at -4200 V, source 187
temperature (TEM) at 400°C and IonSource gases 1 (GS1) and 2 (GS2) were both 20. MS/MS 188
spectra were collected for 2.04 seconds with Q1 resolution set to low and Q3 resolution set to 189
unit. All data were collected using Analyst 1.5.2 (AB Sciex, Foster City, CA). For the detection 190
of nucleotide sugars separated by reverse phase ion pair chromatography, an API 2000™ 191
LC/MS/MS system (AB Sciex, Foster City, CA) equipped with a TurboIonSpray ion source was 192
10
used. The system was operated in negative ion mode with Q1 scanning only. The ion spray 193
voltage was set at -4200 V, TEM at 350 °C, GS1 was 30 and GS2 was 6. MS spectra were 194
collected for 2.3 seconds with Q1 set to unit. Q1 scanning data was collected and analyzed with 195
Analyst 1.4.2 (AB Sciex, Foster City, CA). 196
197
Data analysis 198
Nucleotide sugars from plant extracts were quantified by taking the integrated signal peak area 199
using the Analyst 1.5.2 and MultiQuant™ 2.1 (build 2.1.1296.02.1) software packages (AB 200
Sciex, Foster City, CA). These values were used to calculate the amount of substrate using linear 201
regression from a calibration curve of known nucleotide sugar standards run at the start and end 202
of the analysis period. A mixture of nucleotide sugar standards was used to create the standard 203
curve comprising 2.5, 5, 10, 25, 50, 100, 250, 500 fmol, and 1, 2.5, 5, 10, 25, 100, 250 pmol. 204
Calculations were undertaken using Microsoft Excel (Microsoft Corporation, WA). 205
206
Linearity, repeatability, limit of detection, limit of quantification and recovery 207
To determine linearity, standard mixtures of nucleotide sugars ranging from 2.5 fmol to 250 208
pmol were analyzed to obtain an external calibration curve for each compound. Repeatability 209
was determined by analyzing three different amounts of each standard compound (n=3). The 210
coefficient of variation (CV%) is 100 × standard deviation/mean for the retention times of three 211
different concentration levels of standards. Limit of detection (LOD) and limit of quantification 212
(LOQ) were calculated for signal/noise ratios of 3:1 and 10:1, respectively, that had been 213
11
determined during the measurements of linearity. Recovery was determined by the addition of 214
each standard to plant extracts both before metabolite extraction and after metabolite extraction. 215
This was undertaken using both a physiological amount of each nucleotide sugar (based on a 216
plant extracts) and using three times this amount to test the effect of ion suppression. Recovery 217
was determined with n=4 biological samples for both before and after extraction. 218
12
Results and Discussion 219
Separation and detection of nucleotide sugar standards by ZIC-HILIC and tandem mass 220
spectrometry 221
The biosynthetic pathways of the principle nucleotide sugars incorporated into plant cell walls is 222
largely known (Figure 1) [2; 3; 5]. A major challenge in quantifying plant nucleotide sugars 223
involved in cell wall biosynthesis by LC-MS/MS is to separate structural isomers such as UDP-224
Xyl/UDP-Ara, UDP-Glc/UDP-Gal, UDP-GlcA/UDP-GalA, and GDP-Glc/GDP-Man [12]. Other 225
than GDP-Glc/GDP-Man, the aforementioned nucleotide sugar isomers undergo identical 226
fragmentation, which necessitate their separation with optimized chromatographic conditions. 227
Further complicating this issue, in plants UDP-Arap is converted by UDP-Ara mutases into 228
UDP-Araf prior to its incorporation into matrix polysaccharides and deposition into the cell wall 229
[10]. Prior work investigating a variety of hydrophilic metabolite standards had demonstrated the 230
efficacy of employing HILIC for the separation of nucleotide sugars [17; 21]. Consequently we 231
sought to optimize the ZIC-HILIC LC-MS/MS approach for the separation and quantification of 232
mixtures of nucleotide sugars with a focus on those commonly found in plant tissues. A total of 233
eight of the twelve nucleotide sugar standards were successfully separated by mass and/or 234
retention time (Figure 2). The nucleotide sugars standards UDP-Rha, UDP-Api, CMP-Kdo and 235
GDP-Gal were excluded from this analyses as they were not commercially available. In the case 236
of CMP-Kdo and UDP-Api, both have been reported to be highly labile compounds with short 237
half-lives of 34 minutes at pH7.5 and 25ºC [22] and 97.2 minutes at pH 8.0 and 25ºC [23], 238
respectively. 239
240
13
Chromatographic separation of structural isomers using ZIC-HILIC 241
Despite testing various LC conditions which included varying pH between 3 to 8, ammonium 242
acetate concentrations (10mM or 20mM), column temperatures, gradient slopes, solvent 243
concentrations and flow rates 20 to 50 μL min-1, we were unable to separate the structural 244
isomers UDP-Glc/UDP-Gal or the N-acetylated amino-sugars UDP-N-acetyl-D-galactosamine 245
(UDP-GalNAc)/UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) using ZIC-HILIC 246
chromatography (Figure 2). 247
A previous chromatographic separation technique applied to the analysis of nucleotide sugars 248
which employed porous graphitic carbon (LC-MS) was also unable to separate the UDP-249
GlcNAc/GalNAc isomers [11]. Only the separation procedure employing high performance 250
anion exchange chromatography (LC-MS/MS) [12] has been successful in separating these 251
isomers, although this technique was not successful in separating GDP-Man/Glc. Both UDP-252
GlcNAc and UDP-GalNAc are major substrates for protein glycosylation in eukaryotic systems 253
but are not incorporated into polysaccharide polymers of the cell wall. Although they were 254
indistinguishable when applying the ZIC-HILIC separation approach, plants do not undertake 255
GalNAc O-glycosylation of proteins unless engineered with both a GlcNAc C4-epimerase and a 256
UDP-GalNAc polypeptide N-acetylgalactosaminyltransferases [24; 25]. As a consequence, the 257
UDP-GlcNAc/GalNAc signal measured from plant samples can be assumed to essentially 258
constitute UDP-GlcNAc. 259
The chromatographic separation of the structural isomers UDP-Glc/Gal by ZIC-HILIC, was also 260
not successful under conditions tested. In contrast, both recent approaches employing porous 261
graphitic carbon (LC-MS) [11] or high performance anion exchange chromatography (LC-262
14
MS/MS) [12] have successfully distinguished these isomers. Nonetheless, we could demonstrate 263
using standards, that the calibration curves for UDP-Glc and UDP-Gal are almost identical when 264
using ZIC-HILIC and LC-MS/MS (Figure S1). Consequently, if the abundance and presence of 265
UDP-Glc and UDP-Gal is required, samples can be analyzed using a complimentary technique, 266
such as a reverse phase LC-MS/MS method with an ion pairing agent [26] to determine their 267
respective intensity ratios. This value can then be used to estimate their abundances in samples 268
analyzed by ZIC-HILIC as is demonstrated below. 269
270
Performance of ZIC-HILIC coupled to mass spectrometry 271
Different concentrations of standards were used to obtain an external calibration curve for each 272
available nucleotide sugar. Coupling the ZIC-HILIC separation technique to mass spectrometry 273
provided good sensitivity of nucleotide sugar standards at low femtomole levels. For most 274
nucleotide sugars, their calibration curves demonstrated linearity across a wide range of 275
concentrations from low fmol to upper levels of 100 to 250 pmol resulting in R2 > 0.98 for all 276
standards (Table 2). The exceptions were UDP-Arap and UDP-Araf which had narrower linear 277
ranges that extended to 25 pmol and UDP-Glc and UDP-Gal which extended to 50 pmol. 278
The limits of detection (LOD) varied from 2.5 and 5 fmol and the limits of quantification (LOQ) 279
were between 5 and 20 fmol (Table 2). This was a considerable improvement on the high 280
performance anion exchange chromatography separation technique (LC-MS/MS) examining 281
plant nucleotide sugars which report LODs of 0.3 to 34 pmol and LOQs of 1 to 111 pmol [12]. 282
LOD measurements with the porous graphitic carbon LC-MS method [11] was only performed 283
on four nucleotide sugars; UDP-GlcNAc, UDP-Glc, GDP-Fuc, and UDP-GlcA using ~25 fmol 284
15
in each case. This was because the authors focused on the separation of nucleotides and 285
nucleotide sugars on PGC columns, and not on the conditions for their detection [11]. These 286
significance differences in LOD and LOQ for ZIC-HILIC highlight the unique compatibility of 287
this separation technique when coupled to mass spectrometry. 288
The repeatability of the ZIC-HILIC method was examined by running triplicates for three 289
different concentrations of standard mixtures (Table 2). The coefficients of variation (CV%) of 290
retention times for nucleotide sugars were calculated to be less than 8.34%, 3.48% and 3.90%, 291
for 100 fmol, 1 pmol and 10 pmol of standard mixtures, respectively. Overall, these numbers 292
indicated repeatability of the LC-MS/MS method with the nucleotide sugar standard mixtures. 293
An assessment of sample recovery and matrix effects using ZIC-HILIC were explored using 294
standard additions of nucleotide sugars to plant leaf extracts. The amount of standard applied 295
was initially determined from the physiological values measured in plant samples (Table 3). 296
Sample recovery for the majority of nucleotide sugars was within an acceptable range with 297
recovery rates ranging from ca. 84 to 110% (standard before extraction, Table 3). Only the 298
standard addition of 3-fold physiological amounts of UDP-GlcNAc/GalNAc (120 pmol) and 299
UDP-Glc/Gal (2800 pmol) gave a less than adequate recovery rates with values of 78.9 and 75.4 300
%, respectively. Overall, these data also indicate minimal matrix effects during the ZIC-HILIC 301
technique. This was further explored through standard additions after extraction (Table 3). 302
Generally, the matrix had a minor impact on detection and quantification even with very high 303
amounts of standard (3-fold) which was also used to assess ion suppression. Values for the 304
majority of nucleotide sugar standards were in the range of ca. 82 to 109% with the exception of 305
UDP-GlcNAc/GalNAc (40 pmol and 120 pmol) with values 68.4 and 79.6 % and UDP-Xyl (35 306
pmol) with a value of 77.8 %. Taken together, high amounts of UDP-GlcNAc/GalNAc in a 307
16
sample are likely to be affected by the sample matrix or result in ion suppression when using the 308
ZIC-HILIC approach. Overall, these results demonstrate both the reliability of the nucleotide 309
sugar extraction procedure on plant material and the minimal effects of the plant matrix on the 310
separation and quantification of nucleotide sugars. 311
312
Nucleotide sugar analysis of leaf extracts with differing cell wall compositions 313
While cell walls of the model dicot plant, Arabidopsis and model monocot, rice (Oryza sativa) 314
are composed mainly of the β-1,4-glucan polymer cellulose they contain different matrix 315
polysaccharide structures [27; 28; 29]. Indeed, comparing the monosaccharide compositions of 316
Arabidopsis and rice leaves showed significantly higher levels of fucose (Fuc), rhamnose (Rha), 317
galactose (Gal), mannose (Man) and galacturonic acid (GalA) in Arabidopsis leaf extracts and a 318
significantly higher level of glucose (Glc) and xylose (Xyl) in rice leaf samples (Figure 3). These 319
monosaccharide composition differences reflect the high amount of pectin in Arabidopsis leaves 320
[30] and the presence of arabinoxylan and mixed linkage glucan in rice leaves [31]. 321
Since nucleotide sugars are the precursors for these cell wall polysaccharides, we applied the 322
highly sensitive ZIC-HILIC-based LC-MS/MS method to compare levels of nucleotide sugar 323
precursors between leaves of Arabidopsis and rice (Figure 4). Only small amounts of plant 324
sample (~500 µg FW) were needed in our analysis of nucleotide sugars, as higher amounts were 325
outside the linear range (of the calibration curve) necessary for quantification. This can be 326
especially beneficial when plant samples of interest are only available in low quantities. A solid 327
phase extraction (SPE) technique employing microporous amorphous carbon (ENVI-Carb) has 328
previously been shown to extract nucleotide sugars from bacterial lysates [20]. We have now 329
17
shown its suitability for plant extracts using standard additions and demonstrated excellent 330
recovery rates (Table 3). The inclusion of this clean-up step is essential for the reliable analysis 331
of these compounds from plant material using the ZIC-HILIC approach. Consequently, we used 332
this SPE method to enrich nucleotide sugars from plant leaf extracts prior to their analysis by 333
mass spectrometry. 334
The total quantities of nucleotide sugars per mg fresh weight for Arabidopsis and rice leaf 335
samples are outlined in Table 4 and Figure S2. Levels of UDP-Glc and UDP-Gal were calculated 336
from peak area ratios of their separations in three different dilutions of Arabidopsis and rice 337
samples by reverse phase ion pair chromatography LC-MS (Figure S3). Significant differences 338
between Arabidopsis and rice leaves were identified with UDP-Glc (1.19-fold higher in 339
Arabidopsis), UDP-Gal (1.16-fold higher in Arabidopsis), UDP-GalA (3.46-fold higher in 340
Arabidopsis), UDP–Araf (5.09-fold higher in rice), GDP-Fuc (5.64-fold higher in rice) and GDP-341
Glc (2.1-fold higher in rice) varying between the two species. Previous analyses using the porous 342
graphitic carbon LC-MS method had not successfully detected GDP-Glc in Arabidopsis leaves 343
[11].While the high performance anion exchange chromatography (LC-MS/MS) technique was 344
unable to separate GDP-Glc from GDP-Man [12], here the ZIC-HILIC method was sensitive 345
enough to quantify this low-abundant nucleotide sugar in both Arabidopsis and rice leaves 346
(Table 4). Although we observed some cross-talk with GDP-Man (Figure 4), GDP-Glc could be 347
differentiated and quantified from plant extracts. The ZIC-HILIC technique was also capable of 348
consistently detecting a transition for UDP-Rha (549/323 m/z) as a significant peak at 16.9 349
minutes in both Arabidopsis and rice leaf samples (Figure 4). However, this transition still needs 350
to be verified once the standard becomes commercially available. 351
18
This analysis and quantification of cell wall substrates from Arabidopsis and rice leaves and the 352
corresponding composition of their cell walls enables a cursory examination of their relationship. 353
The most dramatic differences in cell wall composition are those of GalA in Arabidopsis leaves 354
and Xyl in rice leaves (Figure 3). No significant differences were observed for UDP-Xyl 355
between Arabidopsis and rice, while UDP-GalA were ca. 3-fold higher in Arabidopsis leaves. 356
Biosynthesis of these two nucleotide sugars diverges at UDP-GlcA (Figure 1), the similar UDP-357
Xyl levels may indicate a default route, with production of UDP-GalA more tightly regulated. 358
Although a recent analysis of metabolic flux of nucleotide sugars in Arabidopsis cell cultures did 359
not indicate differential carbon flow at this point in the pathway [13]. There were also a number 360
of opposite relationships observed, including GDP-Fuc / Fuc content and UDP-Glc / Glc content. 361
In Arabidopsis leaves, Fuc content was significantly higher, but GDP-Fuc levels lower while in 362
rice leaves Glc comprised a higher proportion of the cell wall but the levels of UDP-Glc levels 363
were lower. These examples may reflect demand on these metabolic pools during cell wall 364
biosynthesis. Since GDP-Fuc is synthesized via GDP-Man (Figure 1), which has similar levels in 365
both species, again it’s possible that these data indicate a high degree of regulation at this point 366
in the pathway. 367
368
Reported nucleotide sugar levels in Arabidopsis 369
Concentrations of nucleotide sugars measured in Arabidopsis samples using the ZIC-HILIC 370
technique resulted in some differences to previous analysis techniques. Although the prior 371
application of the high performance anion exchange chromatography technique was applied to a 372
different cell type, namely cell cultures, there was a considerable level of agreement between 373
19
results [12]. Although data were presented as dry weight, a comparison of nucleotide sugar ratios 374
(based on UDP-Glc) between our study indicates that only the level of UDP-Xyl was 375
significantly different (an order of magnitude lower) in the cell cultures when compared to 376
Arabidopsis leaf tissue (this study). In contrast, nucleotide sugar levels isolated from similar 377
material (Arabidopsis leaf tissue) analyzed by porous graphitic carbon LC-MS were all generally 378
an order of magnitude lower (ranging from 2.4 fmol mg FW-1 for UDP-Araf to 280 fmol mg FW-379
1 for UDP-Glc) [11]. When compared to measurements outlined in this study, values were 380
generally in the pmol mg FW-1 range for leaf material. However, the main objective of the 381
porous graphitic carbon LC-MS study was to establish optimal chromatographic conditions for 382
nucleotide and nucleotide sugar separations, with quantitative analysis of biological samples by 383
MS a secondary concern [11]. 384
385
Separation and identification of nucleotide sugar structural isomers in plant samples 386
The analyses of plant samples in this study using the ZIC-HILIC LC-MS/MS procedure was 387
specifically focused on the analysis and quantitation of major nucleotide sugars involved in cell 388
wall biosynthesis. With the exception of UDP-Rha, which is both a major metabolite and a 389
significant component of plant cell walls, we only targeted nucleotide sugars with available 390
metabolic standards. Nonetheless, there are a number of structural isomers with potentially 391
identical transition states that need to be considered when analyzing these data from plant 392
samples. 393
The separation and identification of the structural isomers UDP-Xyl, UDP-Araf and UDP-Arap 394
could be successfully accomplished using ZIC-HILIC and LC-MS/MS. The branched chain five 395
20
carbon sugar apiose is generally minor component of the pectin polymer rhamnogalacturonan II 396
in the cell walls of plants and is likely present as UDP-Api in its activated form [32]. Although 397
UDP-Api is an isomer of UDP-Xyl, UDP-Araf and UDP-Arap, no evidence for this compound 398
was present in our analysis of cell wall material from Arabidopsis or rice (Figure 4). While this 399
could indicate that the technique could not resolve this compound, it should be noted that the 400
half-life of UDP-Api is less than 2 hours at room temperature [23]. The identification and 401
separation of UDP-Api has not been reported in plant samples by any of the recent mass 402
spectrometry-based analysis procedures [11; 12] and may further indicate the labile nature of this 403
compound. 404
The nucleotide sugars GDP-Gal and GDP-L-Gulose (GDP-Gul) are both synthesized from GDP-405
Man and are confirmed (GDP-Gal) or proposed (GDP-Gul) as intermediates in the biosynthesis 406
of ascorbic acid [33]. Together with GDP-Glc, these four nucleotide sugars are structural isomers 407
and consequently may complicate the analyses of complex metabolic samples. No evidence for 408
either GDP-Gal or GDP-Gul could be observed from plant samples of Arabidopsis or rice 409
employing either the 604/424 or 604/362 transitions (Figure 4). Thus, it is possible that under our 410
conditions, ZIC-HILIC chromatography is not able to distinguish these nucleotide sugars. It has 411
been reported that the application of porous graphitic carbon LC-MS was able to separate and 412
identify GDP-Man, GDP-Gul and GDP-Gal in plant extracts from Arabidopsis and tobacco 413
while no GDP-Glc was observed [11]. Although without standards for GDP-Gul, GDP-Gal or 414
GDP-Glc, it is unclear how the authors could distinguish between these isomers and thus as 415
noted in their study, GDP-Gul and GDP-Glc are tentatively assigned. The high performance 416
anion exchange chromatography (LC-MS/MS) technique did not report the presence of GDP-Gal 417
or GDP-Gul [12]. Given the importance of ascorbic acid biosynthesis in plant development [34], 418
21
it is possible that the reported GDP-Glc or GDP-Man peaks identified in these studies (and our 419
own analysis) may also contain GDP-Gal. 420
421
Conclusion 422
We have implemented a method based on ZIC-HILIC and selected reaction monitoring mass 423
spectrometry to separate and quantify nucleotide sugars. The approach is sensitive and could be 424
deployed on most biological material provided nucleotide sugar standards are available for 425
reliable quantitation. Using different quantities of nucleotide sugar standard mixtures, we 426
demonstrated high sensitivity, linear range and repeatability of the method. The extraction 427
procedure and enrichment step showed reproducible recovery rates and resulted in a minimal 428
impact from the sample matrix. Comparative quantitative analyses of Arabidopsis and rice leaf 429
extracts validated its applicability to plant samples by revealing significant differences in 430
concentrations of six out of twelve nucleotide sugars measured. This approach will provide a 431
complementary analytical tool with other recently developed LC-MS/MS methods to study the 432
dynamics of the complex metabolic processes involved in plant cell wall biosynthesis. 433
22
Acknowledgments 434
This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, 435
Office of Biological and Environmental Research, of the U.S. Department of Energy under 436
Contract No. DE-AC02-05CH11231. The substrates UDP-xylose, UDP- arabinopyranose, UDP-437
galacturonic acid were obtained from Carbosource Services (Athens, GA) which is supported in 438
part by NSF-RCN grant # 0090281. 439
23
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27
Table 1. Specific settings used for the analysis of nucleotide sugar standards by selected 527
reaction monitoring (SRM). 528
Compound Precursor Ion
[M-H]-
Product Ion
[M-H]-
Collision
Energy (eV)
UDP-Xyl 535 323 30
UDP-Arap 535 323 30
UDP-Araf 535 323 30
UDP-Glc 565 323 33
UDP-Gal 565 323 33
UDP-GlcA 579 403 30
UDP-GalA 579 403 30
GDP-Fuc 588 442 30
GDP-Glc 604 362 33
GDP-Man 604 442 36
UDP-GlcNAc 606 385 33
UDP-GalNAc 606 385 33
529
Values were determined empirically through infusion of standards into the mass spectrometer. 530
531
28
Table 2. Linearity, repeatability and detection limits of nucleotide sugar standards analyzed by ZIC-HILIC and LC-MS/MS
Compound Retention Time Linear Range Correlation Coefficient Repeatability (CV%) Detection Limit
(minutes) (pmol) (R2) 100 fmol 1 pmol 10 pmol LOD (fmol) LOQ (fmol)
UDP-Glc/Gal 18.00 0.005–50 0.9814 1.92 2.25 1.92 2.5 5
UDP-Arap 18.00 0.005–25 0.9910 1.12 1.39 1.39 2.5 10
UDP-Araf 15.40 0.005–25 0.9980 2.39 3.40 3.90 5 20
UDP-Xyl 17.20 0.005–100 0.9908 8.34 2.53 2.97 2.5 10
UDP-GlcA 21.00 0.02–250 0.9927 0.55 0.55 0.48 5 20
UDP-GalA 20.50 0.05–250 0.9977 1.02 0.74 0.74 5 20
UDP-GlcNAc/GalNAc 15.70 0.01–250 0.9885 2.31 3.48 3.80 2.5 10
GDP-Man 20.90 0.01–250 0.9852 0.28 0.48 0.73 5 20
GDP-Fuc 19.85 0.005–250 0.9957 0.58 1.26 0.77 2.5 10
GDP-Glc 20.40 0.005–100 0.9883 0.85 0.28 0.49 2.5 5
29
Table 3. Recovery of nucleotide sugars analyzed by ZIC-HILIC and LC-MS/MS
Compound Amount
relative to plant samples
Standard addition (pmol)
Standard before extraction % ± stdev
Standard after extraction % ± stdev
1 x 927 110.9 ± 5.0 89.2 ± 0.7 UDP-Glc/Gal (4:1)
3 x 2800 78.9 ± 0.9 82.3 ± 3.6 1 x 54 89.9 ± 5.4 89.2 ± 0.7 UDP-Arap
3 x 162 80.3 ± 2.9 82.3 ± 3.6 - - ND ND UDP-Araf
- - ND ND 1 x 35 99.7 ± 9.2 77.8 ± 2.4 UDP-Xyl
3 x 105 108.3 ± 12.0 91.9 ± 7.6 1 x 33 104.6 ± 6.1 98.9 ± 3.0 UDP-GlcA
3 x 100 110.9 ± 7.9 101.1 ± 11.4 1 x 27 105.0 ± 6.2 98.8 ± 4.5 UDP-GalA
3 x 81 119.4 ± 9.3 98.8 ± 14.4 1 x 40 101.0 ± 28.0 68.4 ± 1.2 UDP-GlcNAc/GalNAc (1:1)
3 x 120 75.4 ± 4.11 79.6 ± 5.2 1 x 14 85.3 ± 0.9 84.3 ± 4.9 GDP-Man
3 x 42 85.6 ± 7.7 92.2 ± 10.8 1 x 1.9 99.7 ± 9.1 97.9 ± 6.7 GDP-Fuc
3 x 5.7 101.1 ± 4.0 108.5 ± 13.9 1 x 10 84.0 ± 10.7 113.5 ± 6.5 GDP-Glc
3 x 30 98.1 ± 6.4 100.7 ± 15.5
Recovery was not determined (ND) for UDP-Araf due to limited availability of the standard. For
UDP-Glc/Gal and UDP-GlcNAc/GalNAc which could not be resolved using ZIC-HILIC,
standard additions were applied using ratios of nucleotide sugars.
30
Table 4. Nucleotide sugar concentrations in leaves from Arabidopsis and rice plants
Nucleotide Sugar Arabidopsis
(pmol mg FW-1)
rice
(pmol mg FW-1) p value
UDP-Glc 41.74 ± 0.82a 35.14 ± 2.23a 0.009*
UDP-Gal 11.82 ± 0.24a 10.17 ± 0.64a 0.014*
UDP-GalA 1.21 ± 0.30 0.35 ± 0.02 0.008*
UDP-GlcA 0.47 ± 0.20 0.17 ± 0.03 0.060
UDP-Araf 0.05 ± 0.02 0.27 ± 0.03 0.001*
UDP-Arap 6.40 ± 0.46 7.80 ± 1.47 0.189
UDP-Xyl 3.84 ± 0.36 3.49 ± 0.72 0.493
UDP-GlcNAc/GalNAc 3.81 ± 0.68 2.59 ± 0.37 0.051
GDP-Man 0.55 ± 0.20 0.71 ± 0.07 0.256
GDP-Fuc 0.11 ± 0.02 0.62 ± 0.14 0.003*
GDP-Glc 0.015 ± 0.006b 0.032 ± 0.003 0.010*
Values are given as the mean ± standard deviation of three biological replicates. a Values were
calculated using ratios outlined in Figure S3. b Amount detected during analysis (~1.5 fmol) was
below limit of quantitation (LOQ) as outlined in Table 2. As calculated by two-tailed Student's t-
Test, p values marked with an asterisk were significantly different (p < 0.05) between plant
species.
31
Figure Legends
Figure 1. Nucleotide sugar biosynthetic pathways in plants.
Schematic pathways for common nucleotide sugar substrates incorporated into plant cell walls.
Nucleotide sugars in black boxes were analyzed in this study. Abbreviations not defined in text:
D-Glc-1-P: D-glucose-1-phosphate, D-Glc-6-P: D-glucose-6-phosphate, D-Fru-6-P: D-fructose-
6-phosphate, D-Man-6-P: D-mannose-6-phosphate, D-Man-1-P: D-mannose-1-phosphate, D-
GlcN-6-P: D-glucosamine-6-phosphate , D-GlcNAc-1-P: N-acetyl-D-glucosamine-1-phosphate,
PPP: pentose phosphate pathway, Kdo-8-P: ketodeoxyoctonate-8-phosphate.
Figure 2. Ion chromatograms of nucleotide sugar standards analyzed by ZIC-HILIC and
LC-MS/MS
A total of 100 fmol of a nucleotide sugar standard mix was separated and quantified using ZIC-
HILIC LC-MS/MS. Total ion chromatogram (TIC) of the mixture of nucleotide sugar standards
is presented in the top panel. The precursor/product ion MS/MS transition for each nucleotide
sugar is indicated in the top right corner of each panel. The LC conditions employed are outlined
in the methods section.
Figure 3. Monosaccharide composition of Arabidopsis and rice leaf material.
Cell wall monosaccharide compositions of rosette leaves from Arabidopsis plants (dark grey
bars, mean ± std dev, n = 3 biological replicates) was compared to leaves of rice plants (light
grey bars, mean ± std dev, n = 3 biological replicates). Sugars marked with an asterisk (*)
32
showed significant differences (p < 0.05) between Arabidopsis and rice leaf samples, calculated
by two-tailed Student's t-Test.
Figure 4. Ion chromatograms of nucleotide sugars from the two different plant samples.
Enriched nucleotide sugars from (A) Arabidopsis and (B) rice leaves were separated and
quantified by ZIC-HILIC LC-MS/MS. Total ion chromatograms (TIC) of (A) Arabidopsis and
(B) rice leaves are presented in the top two panels. The precursor/product ion MS/MS transitions
for each nucleotide sugar are indicated on the right of the chromatograms. The bottom panels
both show a transition (549/323) at 16.9 minutes marked by an asterisk (*) representing UDP-
Rha. This could not be verified because a UDP-Rha standard is not commercially available.
UDP-Glc
UDP-Gal
UDP-GlcA
UDP-Xyl
UDP-Api
UDP-Rha
D-Glc-1-PGDP-Glc
D-Glc-6-P
D-Fru-6-P
D-Man-6-P
D-Man-1-P GDP-Man
GDP-Fuc
UDP-Arap UDP-Araf
UDP-GalA
D-GlcN-6-P D-GlcNAc-1-P
UDP-GlcNAc
UDP-GalNAc
Figure 1.
GDP-Gal
CMP-Kdo
Kdo-8-P
Kdo
PPP
SUCROSE
UDP-Arap
UDP-GalA
TIC
UDP-Glc/Gal
UDP-GlcA
UDP-Araf
UDP-Xyl
UDP-GlcNAc/GalNAc
GDP-Man
GDP-Glc
GDP-Fuc
Transition:
565/323
Transition:
606/385
Transition:
535/323
Transition:
579/403
Transition:
604/424
Transition:
588/442
Transition:
604/362
Time (min)
Rel
ati
ve
Inte
nsi
ties
(%
)
0 42
0
100
0
100
0
100
0
100
0
100
0
100
0
100
0
100
Figure 2
Figure 3
0
10
20
30
40
50
60
70
*Fuc *Rha Ara *Gal *Glc *Xyl *Man *GalA GlcA
Rel
ati
ve
con
ten
t (m
ol%
)
Monosaccharides
Arabidopsis Rice
UDP-Glc/Gal UDP-Glc/Gal
UDP-GalA
UDP-GlcA
UDP-GalAUDP-GlcA
UDP-Arap
UDP-Araf
UDP-XylUDP-Arap
UDP-Araf
UDP-Xyl
UDP-GlcNAc/GalNAc UDP-GlcNAc/GalNAc
GDP-ManGDP-Man
GDP-Fuc GDP-Fuc
A
Transition
565/323
Transition
606/385
Transition
579/403
Transition
535/323
Transition
604/424
Transition
588/442
Transition
549/323
* *
Rel
ati
ve
Inte
nsi
ties
Time (min)
Transition
604/362GDP-Glc GDP-Glc
TIC TICB
Figure 4
