MTBLS4: High-resolution extracted ion chromatography, a new tool for metabolomics and lipidomics using a second-generation orbitrap mass spectrometer    

Albert Koulman , Dietrich Volmer

Study Submission Date: 14-Feb-2012   Study public release date: 14-Feb-2012

Most analytical methods in metabolomics are based on one of two strategies. The first strategy is aimed at specifically analysing a limited number of known metabolites or compound classes. Alternatively, an unbiased approach can be used for profiling as many features as possible in a given metabolome without prior knowledge of the identity of these features. Using high-resolution mass spectrometry with instruments capable of measuring m/z ratios with sufficiently low mass measurement uncertainties and simultaneous high scan speeds, it is possible to combine these two strategies, allowing unbiased profiling of biological samples and targeted analysis of specific compounds at the same time without compromises. Such high mass accuracy and mass resolving power reduces the number of candidate metabolites occupying the same retention time and m/z ratio space to a minimum. In this study, we demonstrate how targeted analysis of phospholipids as well as unbiased profiling is achievable using a benchtop orbitrap instrument after high-speed reversed-phase chromatography. The ability to apply both strategies in one experiment is an important step forward in comprehensive analysis of the metabolome.

Organism(s):
Homo sapiens (Human)

Study Design Description:
  • metabolomic profiling
  • lipipdomics
  • orbitrap
  • open profiling

Publications
High-resolution extracted ion chromatography, a new tool for metabolomics and lipidomics using a second-generation orbitrap mass spectrometer.

Experimental Factors
  • healthy volunteers: [ Accepts Healthy Volunteers Indicator ]
  • blood plasma: [ plasma ringer palm , plasma ringer gluc cer palm , plasma ringer cer , plasma ringer gluc , plasma ringer , plasma ]
Protocol Description
Sample collection Plasma samples were collected from 10 healthy volunteers. For each sample, 200 µL aliquots were taken and diluted with either (a) 100 µL of Ringer solution; (b) 40 µL of Ringer solution and 10 µL of palmitic acid stock solution; (c) 40 µL of Ringer solution and 10 µL of glucose stock; (d) 40 µL of Ringer solution and 10 µL of N-octanoylsphingosine stock; (e) 40 µL of Ringer solution, 10 µL of palmitic acid stock, 10 µL of glucose, 10 µL of N-octanoylsphingosine stock. Also, 250 µL of whole plasma was used, yielding a set of total 60 samples.
Extraction A volume of 250 µL of each sample was added to 1000 µL of cold acetronitrile. This mixture was centrifuged for 10 min at 13 000 rpm and the supernatant was diluted (1:1) with formic acid (0.1%) and transferred to a 96-well plate ready for analysis by high-performance liquid chromatography (HPLC).
Chromatography Chromatographic separations were performed on a 5.0 × 2.1 mm Hypersil Gold 1.9 µm C18 column (Thermo Scientific, Runcorn, UK) using an Accela U-HPLC system (Thermo Scientific, Hemel Hempstead, UK). The column was maintained at 45°C. A binary mobile phase system was used where A=formic acid (0.1%) and B=acetonitrile/isopropyl alcohol (1:1) containing formic acid (0.1%). The mobile phase program at an initial hold (0.0–0.5 min) at 5% B followed by a linear gradient 5–50% B (0.5–5.0 min), then 50–95% B (5.0–5.5 min); the conditions were then held at 95% B (5.5–6.5 min) and returned to the initial conditions (6.5–10.0 min). The total analysis duration was 10 min at a flow rate of 0.25 mL/min. The 60 samples were run in randomised order and were injected using an injection volume of 10 µL.
Mass spectrometry Mass spectrometry was performed on an Exactive orbitrap mass spectrometer (Thermo Scientific, Hemel Hempstead, UK) operating in positive ion mode. The heated electrospray (HESI-II) source was used. The sheath gas was set to 20 (arbitrary units) at a temperature of 200°C, the aux gas set to 10 (arbitrary units) and the capillary temperature set to 250°C. The capillary voltage and spray voltage were set to 51 V and 4.2 kV, respectively. The instrument was operated in full scan mode from m/z 150–1000 at 50 000 resolving power. The data acquisition rate was 2 Hz. The mass spectrometer was mass calibrated just prior to starting the sequence of 240 injections. All data was acquired using lock mass calibration (m/z 214.0896).
Data transformation The raw files were converted into NetCDF files using the Thermo software package Xcalibur. The converted files were imported into MZmine18 and peaks were detected using the following settings: noise level =30000.0; mass resolution =30000; peak model function =‘Savitzky-Golay’; min time span =7.0; m/z tolerance =0.0020. The resulting peak lists were aligned using m/z tolerance =0.0025; retention time tolerance =10.0%. The resulting peak list was exported as a CSV file and imported into DANTE where missing data was imputed using the k Nearest Neighbour method.
Metabolite identification For the targeted analysis of 50 specific phospholipids (see Table 2), the theoretical exact masses were used with 4 significant figures with a scan width of ±2.5 ppm. The resulting extracted ion chromatograms were integrated and the area-under-the-curve (AUC) was used for relative quantitation. The values were imported into the Dante software, where missing data was imputed using the k Nearest Neighbour method.

Technology - Platform: mass spectrometry - metabolite profiling - Exactive (Thermo Scientific)
Source blood plasma healthy volunteers
MRC_Run1_10uL_C11 plasma ringer palm Accepts Healthy Volunteers Indicator
MRC_Run1_10uL_C48 plasma ringer gluc cer palm Accepts Healthy Volunteers Indicator
MRC_Run1_10uL_C47 plasma ringer gluc cer palm Accepts Healthy Volunteers Indicator
MRC_Run1_10uL_C05 plasma ringer palm Accepts Healthy Volunteers Indicator
MRC_Run1_10uL_C54 plasma ringer gluc cer palm Accepts Healthy Volunteers Indicator
MRC_Run1_10uL_C38 plasma ringer cer Accepts Healthy Volunteers Indicator
MRC_Run1_10uL_C25 plasma ringer gluc Accepts Healthy Volunteers Indicator
MRC_Run1_10uL_C01 plasma ringer palm Accepts Healthy Volunteers Indicator
MRC_Run1_10uL_C26 plasma ringer gluc Accepts Healthy Volunteers Indicator
MRC_Run1_10uL_C37 plasma ringer palm Accepts Healthy Volunteers Indicator
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PC(O-38:7) (CHEBI:64539) C46H80NO7P 790.5737 7.32 C[N+](C)(C)CCOP(=O)([O-])OCC(CO)O InChI=1S/C8H20NO6P/c1-9(2,3)4-5-14-16(12,13)15-7-8(11)6-10/h8,10-11H,4-7H2,1-3H3/t8-/m1/s1 7939 NF NF 7809 706915 502607 2911509 1892310 255938 648918 790257 251296 640285 2452485 568824 492880 99773 156099 304825 1084204 334504 228480 307309 425183 366398 481920 273986 456009 1260723 621558 1085495 203507 68264 638211 544520 747365 1132093 1157802 572676 626757 370241 128644 188159 136538 256527 572835 193180 490165 834398 279841 423796 489509 157684 394326 369740 41406 152187 106053 214503 382181
Description Formula m/z Retention time Smiles Inchi MRC_Run1_10uL_C01 MRC_Run1_10uL_C02 MRC_Run1_10uL_C03 MRC_Run1_10uL_C04 MRC_Run1_10uL_C05 MRC_Run1_10uL_C06 MRC_Run1_10uL_C07 MRC_Run1_10uL_C08 MRC_Run1_10uL_C09 MRC_Run1_10uL_C10 MRC_Run1_10uL_C11 MRC_Run1_10uL_C12 MRC_Run1_10uL_C13 MRC_Run1_10uL_C14 MRC_Run1_10uL_C15 MRC_Run1_10uL_C16 MRC_Run1_10uL_C17 MRC_Run1_10uL_C18 MRC_Run1_10uL_C19 MRC_Run1_10uL_C20 MRC_Run1_10uL_C21 MRC_Run1_10uL_C22 MRC_Run1_10uL_C23 MRC_Run1_10uL_C24 MRC_Run1_10uL_C25 MRC_Run1_10uL_C26 MRC_Run1_10uL_C27 MRC_Run1_10uL_C28 MRC_Run1_10uL_C29 MRC_Run1_10uL_C30 MRC_Run1_10uL_C31 MRC_Run1_10uL_C32 MRC_Run1_10uL_C33 MRC_Run1_10uL_C34 MRC_Run1_10uL_C35 MRC_Run1_10uL_C36 MRC_Run1_10uL_C37 MRC_Run1_10uL_C38 MRC_Run1_10uL_C39 MRC_Run1_10uL_C40 MRC_Run1_10uL_C41 MRC_Run1_10uL_C42 MRC_Run1_10uL_C43 MRC_Run1_10uL_C44 MRC_Run1_10uL_C45 MRC_Run1_10uL_C46 MRC_Run1_10uL_C47 MRC_Run1_10uL_C48 MRC_Run1_10uL_C49 MRC_Run1_10uL_C50 MRC_Run1_10uL_C51 MRC_Run1_10uL_C52 MRC_Run1_10uL_C53 MRC_Run1_10uL_C54 MRC_Run1_10uL_C55 MRC_Run1_10uL_C56 MRC_Run1_10uL_C57 MRC_Run1_10uL_C58 MRC_Run1_10uL_C59 MRC_Run1_10uL_C60
lysoPC(14:0) (CHEBI:64483) C8H20NO6P 468.3087 6.62 C[N+](C)(C)CCOP(=O)([O-])OCC(CO)O InChI=1S/C8H20NO6P/c1-9(2,3)4-5-14-16(12,13)15-7-8(11)6-10/h8,10-11H,4-7H2,1-3H3/t8-/m1/s1 8323799 9848204 50496761 17499387 6737567 6803512 3830695 11199567 7559265 6754102 6943806 8676238 10380331 7011717 6987209 6930342 6348159 7490484 5363730 8788597 12595544 7348862 6748154 6913054 6076061 6232689 5175142 3649854 5231908 7088069 5909586 6135553 4883983 6571399 5123207 6735480 10559689 1944974 9169933 3719403 9193726 5541422 13217889 5823877 10500623 5825086 5504368 3983213 10304518 4200401 3310263 4974280 3703736 9646713 7360257 6340342 18522744 5658177 5094902 5790359
lysoPC(16:0) (CHEBI:64563) C24H50NO7P 496.3398 6.89 C[N+](C)(C)CCOP(=O)([O-])OCC(CO)O InChI=1S/C8H20NO6P/c1-9(2,3)4-5-14-16(12,13)15-7-8(11)6-10/h8,10-11H,4-7H2,1-3H3/t8-/m1/s1 518398686 494322343 364886691 540451059 415949574 360081984 220081700 372937222 370289646 300494391 228293425 349114427 444093870 388711573 338586688 395825624 252753597 458995479 344250445 439679657 413540253 368916469 375154367 401354551 300649999 416456184 402211614 236069502 241053966 318097705 361679143 345407637 361779334 252131288 224888130 305429426 377773517 136505708 349131488 261307683 329578632 313931066 352595356 351669389 392794258 250463345 336046883 216484772 449743239 313947325 207526484 341609617 257933520 397347845 432730151 373073234 425406377 316889110 359422776 338459959
lysoPC(16:1) (CHEBI:64560) C24H48NO7P 494.3239 6.67 CCCCCCC=CCCCCCCCC(=O)OCC(COP(=O)([O-])OCC[N+](C)(C)C)O InChI=1S/C24H48NO7P/c1-5-6-7-8-9-10-11-12-13-14-15-16-17-18-24(27)30-21-23(26)22-32-33(28,29)31-20-19-25(2,3)4/h10-11,23,26H,5-9,12-22H2,1-4H3/b11-10-/t23-/m1/s1 18707527 14682208 28213923 26558836 15261685 13228490 5410024 7784547 15536941 10978708 13668405 19465802 25117254 17854981 15733772 17311482 11488603 16508546 9057547 19599215 8707673 11971792 14279598 14084245 9659521 9218238 11563797 5704647 9362045 11947696 13954023 8654565 10326701 12525023 9466083 14860755 24724367 2941461 21550125 5908054 6221883 11608880 11398032 18851109 7450006 10562952 9889637 7461883 26506068 7598268 5104270 11230044 6044700 21649670 19073657 10086795 15710583 8922330 10357574 12237249
lysoPC(18:0) (CHEBI:64561) C26H54NO7P 524.3708 7.22 CCCCCCCCCCCCCCCCOCC(COP(=O)([O-])OCC[N+](C)(C)C)OC(=O)C InChI=1S/C26H54NO7P/c1-6-7-8-9-10-11-12-13-14-15-16-17-18-19-21-31-23-26(34-25(2)28)24-33-35(29,30)32-22-20-27(3,4)5/h26H,6-24H2,1-5H3/t26-/m1/s1 237823559 306284041 170468346 160204084 156038598 130456236 47270501 102446228 132285266 90786251 63703029 122569504 141921320 128008519 139870167 143875238 74957057 127809678 126115879 156034504 96652745 109471324 142424387 183468369 85788389 131279062 133248953 49399782 66279267 86357865 109456304 123815304 107480275 65613801 51367763 88494502 109911435 30303148 113873010 54056624 96414080 107419269 111495371 113730388 118079979 60954636 106598815 39012601 155286992 103428606 52505460 120952837 55191009 112482876 117710915 127118780 133080252 78451982 93953100 119192861
lysoPC(18:1) (CHEBI:64566) C26H52NO7P 522.3554 6.93 C[N+](C)(C)CCOP(=O)([O-])OCC(CO)O InChI=1S/C8H20NO6P/c1-9(2,3)4-5-14-16(12,13)15-7-8(11)6-10/h8,10-11H,4-7H2,1-3H3/t8-/m1/s1 142339702 119834940 103453260 202795630 101638944 95912117 40452943 72520170 96777728 107218567 64420880 126045776 155321249 97264604 89969369 95530057 78932435 123791113 70543017 150765480 83332198 115223446 92194324 103674278 91429268 84293496 96648974 40098962 65447938 91648281 72210564 66235179 75366177 74660641 71594100 90097144 142222438 25295429 119172704 45753960 58841460 69493351 71642212 73903750 70712193 77828355 87125426 39833726 116837398 74679831 36299818 83155640 47502984 117055489 95686164 77641922 84435483 98129799 78467594 83591133
lysoPC(18:2) (CHEBI:64549) C26H50NO7P 520.3392 6.72 CCCCCC=CCC=CCCCCCCCC(=O)OCC(COP(=O)([O-])OCC[N+](C)(C)C)O InChI=1S/C26H50NO7P/c1-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-20-26(29)32-23-25(28)24-34-35(30,31)33-22-21-27(2,3)4/h9-10,12-13,25,28H,5-8,11,14-24H2,1-4H3/b10-9-,13-12-/t25-/m1/s1 167440626 173047147 171626387 412261690 201136397 109590285 68438794 175916589 202762857 261518776 223146679 282080231 414475600 253598356 223436918 249138537 290091749 216102571 119153480 313010844 190780568 308899478 119022172 186041908 244375204 114543122 174879126 72250157 162516769 192572043 168694480 105156527 133216313 218125573 157786341 353823877 363642538 34515304 317897821 82499241 145332821 141533187 234666846 217255656 169709590 202830468 127507619 89992506 223133240 104891813 64738152 165661424 77506843 160028757 224880604 128943221 306436762 227033607 125853925 109133779
lysoPC(18:3) (CHEBI:64565) C26H48NO7P 518.3243 6.59 C[N+](C)(C)CCOP(=O)([O-])OCC(CO)O InChI=1S/C8H20NO6P/c1-9(2,3)4-5-14-16(12,13)15-7-8(11)6-10/h8,10-11H,4-7H2,1-3H3/t8-/m1/s1 3850075 3410581 17828129 10747257 3572006 3445236 953168 3240166 2898446 4474729 2886811 5217325 4826299 3190159 3064880 3296996 4015763 4017263 2002068 4579512 3747840 4749370 3378931 3140325 3883001 2057524 2804768 918918 2122342 3185111 1965582 1762718 1783711 2476675 2278358 3961406 5352452 564651 4609670 945362 2614529 2012566 3304365 2307016 3081914 3132319 2762220 882335 4668600 2044857 794602 2648538 858013 4258634 2716397 2183978 4976785 3680155 1847026 2989369
lysoPC(20:3) (CHEBI:64481) 546.3552 6.8 11276935 6393776 4985923 26944614 7720202 6813838 2885784 4861439 4350407 8123313 7363198 15275477 20156706 8265978 4751106 7984959 6206224 7963836 4417619 18569325 5456070 9892927 7459459 5854857 7889931 4958809 7436548 3175111 6917257 9045247 4752067 4075555 5651553 7776221 6914942 8691332 16646112 1311743 14707873 3737599 4018905 5510348 4665653 6468804 4754204 9172360 4195645 2695175 11082555 3793644 2848766 6104368 3404513 8149646 6774103 4849441 6308385 8214911 5243919 7067417
lysoPC(20:4) (CHEBI:64568) C28H50NO7P 544.3398 6.69 C[N+](C)(C)CCOP(=O)([O-])OCC(CO)O InChI=1S/C8H20NO6P/c1-9(2,3)4-5-14-16(12,13)15-7-8(11)6-10/h8,10-11H,4-7H2,1-3H3/t8-/m1/s1 23347367 37417907 27133890 38448589 29829068 16092992 9680755 15784174 25992896 23566989 21974193 28642028 38180457 36275353 26740897 36265263 25579638 32312587 24196013 29159666 18343914 26009761 17467304 22180710 20925471 23989659 24283243 10394751 14933083 19252209 20950938 22338862 14918884 21220608 15038856 31361066 36835717 7311369 33244089 11029800 13125807 17162228 24033581 27804725 15303970 18524694 16156748 14469322 34907106 12294986 9266939 24144665 10777899 26311369 30404084 27070371 32376697 19126645 15155191 15590168
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