Electronic Supplementary Information Method of coupling ... · Figure S-1: MS2 CID of a side reaction product at m/z 876 from UV irradiation of solution ... ion PIS m/z 153.1 for

S-1

Electronic Supplementary Information

Method of coupling Paternò-Büchi reaction with direct infusion ESI-MS/MS

for locating C=C bond in glycerophospholipids

Craig A. Stinson and Yu Xia

Department of Chemistry, Purdue University, West Lafayette, IN, 47907-2084, USA

Electronic Supplementary Material (ESI) for Analyst.This journal is © The Royal Society of Chemistry 2016

S-2

Experimental procedures

Solutions. Stock solutions of lipids and yeast polar extract were transferred to glass vials with

Teflon lined caps and stored at -20 °C. 5 µL of stock solution was transferred to an Eppendorf

tube containing 95 µL isopropanol using a stainless steel tip glass syringe. From this solution 1-

7.5 µL was further diluted to a 1 mL working solution. Solvent ratios were reported as v/v for all

working solutions. Unless otherwise specified, solutions were purged with N2 prior to analysis to

remove dissolved oxygen. For N2 purging of solutions Eppendorf tubes containing working lipid

solutions were covered with parafilm (Parafilm “M”; Bemis Company, Inc.; Oshkosh, WI, USA)

and a stainless steel needle (Precision Glide Needle; 18 gauge, 1.5” length; BD; Franklin Lakes,

NJ, USA) was inserted to deliver N2 at a bubble frequency of ~ 5 Hz for 15 min.

Mass Spectrometry. All experiments were performed on a triple quadrupole / linear ion trap

(LIT) hybrid mass spectrometer (4000 QTRAP; Applied Biosystems / Sciex; Toronto, Canada).

Data was collected and processed with Analyst software (1.5.2; Applied Biosystems / Sciex;

Toronto, Canada). Neutral loss and precursor ion scans were performed in triple quadrupole

mode.1 For all LIT scans ions were collected and scanned out of Q3 by mass selective axial

ejection (MSAE).2 MS2 beam-type CID consisted of precursor ion selection in Q1, ion

acceleration into q2 collision cell, followed by fragment ion collection and subsequent mass

analysis in Q3. MS3 CID involved precursor ion selection in Q1, ion acceleration into q2 collision

cell, fragment ion collection in the LIT, fragment ion isolation, ion secular frequency resonance

excitation, ion cooling step, followed by MSAE of fragment ions.3 For low abundance ions the

“Q0 trapping” function was activated to increase sensitivity.4 Extracted ion chromatograms

(XICs) were obtained by peak integration of ± 0.5 Da from peak centroid. The typical instrument

settings were: curtain gas, 5 psi; declustering potential, ±100 V; and 1000 Da/s scan time unless

S-3

otherwise specified. MS1 spectra typically employed a 20 ms LIT fill time and were averaged over

30-60 spectra while tandem MS spectra were averaged for at least 120 spectra with LIT fill times

of up to 2 s.

Experimental Data

Figure S-1: MS2 CID of a side reaction product at m/z 876 from UV irradiation of solution

containing PC 16:0/18:1(9Z) from Figure 1b of the main text.

200 300 400 500 600 700 800 900

m/z

x 5.0184.1

858.7876.7

760.6650.5 840.6

478.3 778.6

708.5690.5

606.4

734.6716.5

676.5

100

Re

l. In

t. (

%)

M+116 Da650.5

676.5

PC 16:0/18:1(9Z)

S-4

Continuous UV exposure micro flow reactor for PB-ESI-MS/MS.

Figure S-2: (a) Drawing of a continuous UV exposure reactor used to effect the PB reaction prior

to ESI MS. (b) MS1 reaction spectra showing 9.5 s continuous UV exposure of a solution

containing N2 purged 5 µM PC 16:0/18:1(9Z) in 7:3 acetone: H2O 1% acetic acid using the

experimental setup shown in (a). The GP precursor and PB product are at m/z 760.6 and 818.7,

respectively. (c) MS1 reaction spectrum where the same sample was exposed to 9 s discrete UV

exposure using the experimental setup as shown in Figure 1 of the main text.

Detection limit for PC 16:0/18:1 (9Z)

Figure S-3: MS/MS spectra of the PB reaction product (m/z 818.6) of 5 nM PC 16:0/18:1(9Z)

dissolved in 7:3 acetone: H2O 1% acetic acid to determine the limit of detection of C=C diagnostic

ions at m/z 650.5 and 676.5 (S:N > 3) . UV exposure was 6 s using the continuous UV exposure

reaction system from SI Figure 1a. The MS/MS spectra were averaged for 10 minutes with a 2

second LIT fill time.

a)0.5 cm

LAMP

1.9 cm

UV transparent

fused silica

ESI

syringe

pump

m/z

b)

100 300 500 700 900

818.7760.6

876.7

9.5 s

continuous

exposure

100

Rel. Int.

(%

)

c)

9 s

discrete

exposure

100 300 500 700 900

818.6

760.6

876.6

100

Rel. Int.

(%

)

m/z

100 200 300 400 500 600 700 800

m/z

818.6

481.3184.0 650.5 800.6

676.5

Rel. Int.

(%

)

100

[M+58]+

S-5

Analysis of Yeast Polar Lipid Extract

Procedures. For the yeast polar extract analysis 20 µL of commercially purchased stock (25

mg/mL CHCl3) was dissolved to 1 mL in 7:3 acetone: H2O 1% acetic acid or NH4OH for a final

concentration of 0.5 mg/mL. Prior to direct injection ESI MS/MS the solution was N2 purged as

described in the Experimental Section. Triple quadrupole scans identified PL class and cumulative

number of fatty acyl carbons and degree of saturation. Precursor ion scans (PIS) and neutral loss

scans (NLS) were all implemented at 6 s scan speed. Negative ionization beam-type CID LIT

product ion scans determined the individual fatty acyl chain lengths and degree of saturation. The

PB reaction was implemented using the continuous reaction setup as shown in SI Figure S1a at 5

µL/min and UV exposure of 6-8 s. PB reaction products (+58 Da from precursor ion) for all PL

classes (excluding PC) were subjected to tandem MS to detect C=C diagnostic ions. Ion trap CID

was performed on beam-type CID fragment ions displaying a +58 Da shift from the fatty acyl

fragment ion (i.e. m/z 339.3 and/or 311.3 for fatty acyl m/z 281.3 and 253.3, respectively). PC PB

product ions were subjected to beam-type CID in positive ionization and C=C location was

assigned based on fragment ions and the knowledge of fatty acyl composition from MS/MS beam-

type CID of [M-15]- PC ions.

S-6

Table S-1: Complete list of unsaturated GPs analyzed in the polar yeast extract, including

ionization mode and monoisotopic m/z. Triple quadrupole scans performed to identify GP head

group include neutral loss (NLS) and precursor ion (PIS) scans.

Phospholipid Neutral mass

Ionization Mode

Phospholipid

Monoisotopic m/z

Quadrupole

scan /

headgroup

identifier

PI 16:0_16:1(9) 808.5 (-) 807.5 PI, m/z 241.1

PI 16:1(9)_16:1(9) 806.5 (-) 805.5

PI 16:0_18:1(9) 836.5 (-) 835.5

PI 18:0_16:1(9) 836.5 (-) 835.5

PI 16:1(9)_18:1(9) 834.5 (-) 833.5

PI 18:0_18:1(9) 864.6 (-) 863.6

PI 18:1(9) _18:1(9) 862.6 (-) 861.5

LPI 18:1(9) 598.3 (-) 597.3

LPI 16:1(9) 570.3 (-) 569.3

PC 16:1(9)_16:1(9) 729.5 (+) 730.5 PI, m/z 184.1

PC 16:0_16:1(9) 731.6 (+) 732.6

PC 16:1(9)_18:1(9) 757.6 (+) 758.6

PC 16:0_18:1(9) 759.6 (+) 760.6

PC 18:0_16:1(9) 759.6 (+) 760.6

PC 18:1(9)_18:1(9) 785.6 (+) 786.6

LPC 18:1(9) 522.4 (+) 522.4

LPC 16:1(9) 493.3 (+) 494.3

PE 18:0_16:1(9) 717.5 (+) 718.5 NL, 141.0 Da

PE 16:0_18:1(9) 717.5 (+) 718.5

PE 16:1(9)_16:1(9) 715.5 (+) 716.5

PE 16:1(9)_18:1(9) 715.5 (+) 716.5

LPE 18:1(9) 479.3 (+) 480.3

LPE 16:1(9) 451.3 (+) 452.3

PS 16:1(9)_16:1(9) 703.4 (-) 702.4 NL, 87.1 Da

PS 16:0_16:1(9) 705.5 (-) 704.5

PS 18:0_16:1(9) 733.5 (-) 732.5

PS 16:0_18:1(9) 733.5 (-) 732.5

PS 16:1(9)_18:1(9) 731.5 (-) 730.5

LPS 18:1(9) 523.3 (-) 522.3

LPS 16:1(9) 495.3 (-) 494.3

PA 16:1(9)_16:1(9) 644.4 (-) 643.4 PI, m/z 153.1

PA 16:0_18:1(9) 646.5 (-) 673.5

PA 16:0_16:1(9) 646.5 (-) 645.4

PA 16:1(9)_18:1(9) 644.4 (-) 671.5

LPA 18:1(9) 436.3 (-) 435.3

S-7

Figure S-4: Triple quadrupole neutral loss scans (NLS) and precursor ion scans (PIS) for

determining GP head-group applied to direct injection of yeast polar lipid extract. a) Negative

ion PIS m/z 241.1 for PI at CE = 45. b) Positive ion PIS m/z 184.1 for PC at CE = 35. c) Positive

ion NLS 141 Da for PE at CE = 30. d) Negative ion NLS 87 Da for PS at CE = 35. e) Negative

ion PIS m/z 153.1 for PA/PG at CE = 35.

9.9e4

400 500 600 700 800

716.5

688.4

480.2452.2690.5718.6

482.2 744.7662.3

LPE 16:1 LPE 18:1

PE 16:1_16:1

PE 16:0_16:1

PE 16:1_18:1

PE 16:0_18:1

PE 18:1_18:1PE 14:0_16:1

cps

Pos. ion NLS 141 Da

CE = 30

c)

3.4e4

500 600 700 800 900

835.6

807.6

597.3833.7

863.7571.3 805.6599.4569.1 659.6631.4 753.6

821.6861.6779.6

LPI 16:1

LPI 18:1

PI 16:0_16:1

PI 16:1_16:1

PI 16:0_18:1 PI 16:1_18:1

PI 18:0_18:1 cps

PI 18:1_18:1

Neg. ion PIS m/z 241.1

CE = 45

a)

Pos. ion PIS m/z 184.1

CE = 35

3.0e5

440 500 560 620 680 740 800

494.4

522.4

730.6758.7

732.7760.7440.3 704.6676.6468.4 786.8

PC 18:1

PC 16:1

PC 12:0_16:1

PC 14:0_16:1

PC 16:1_16:1PC 16:0_16:1

PC 16:0_18:1

PC 16:1_18:1

PC 18:1_18:1

PC 18:0_18:1

cps

788.7

b)

1.5e4

450 550 650 750

758.6

760.6522.4

732.6494.4 730.6496.4 524.4

LPS 18:1

LPS 16:1 PS 16:1_16:1

PS 16:0_16:1

PS 16:1_18:1

PS 16:0_18:1

PS 18:1_18:1

cps

786.5

Neg. ion NLS 87 Da

CE = 35

d)

1.8e5

400 500 600 700

407.3409.4

435.3

671.6643.6 645.5 673.6

522.4597.4

518.4571.3449.0

554.4490.2

462.9

703.6

PA 16:0_18:1

PA 16:1_18:1PA 16:1_16:1

PA 16:0_16:1

LPA 18:1

LPA 16:1

PA 18:0_18:1

cps

Neg. ion PIS m/z 153.1

CE = 35

e)

m/z

S-8

Figure S-5: Positive and negative ionization MS1 spectra for the yeast polar lipid extract before

and during application of UV using the experimental setup shown in SI Figure S1a. a) Positive

ionization MS1 spectra before UV and (b) during UV (PB product m/z in red). c) Negative

ionization MS1 spectra before UV and (d) during UV (PB product m/z in red).

400 500 600 700 800 900

758.6730.6

732.6 760.7494.4522.5796.6768.6532.3 560.4

875.7

716.6704.7

577.5

pos. ion. before UV100

Rel. Int. (

%)

a)

400 500 600 700 800 900

835.6

807.6863.6

671.5643.4754.6597.3490.2435.3

571.2409.3 465.3

1000

c)

neg. ion. before UV

m/z

400 500 600 700 800 900 1000

835.6

893.6807.5 917.5863.6

891.6

921.6947.5703.4

788.5

437.3 528.2 729.4478.0

790.5

d)

neg. ion. during UV

400 500 600 700 800 900

758.6

788.6760.6

816.6

494.5 522.5

732.6

844.6776.6

870.6716.6

552.3 688.6 746.6580.3

pos. ion. during UV100 730.6

b)

Re

l. Int. (

%)

Re

l. Int. (

%)

100

100

Rel. Int. (

%)

S-9

Figure S-6: Proposed structure, fatty acyl structural determination, and PB diagnostic ions for PI

18:0_18:1(9) from direct injection of yeast polar lipid extract. a) Beam-type CID of m/z 863.3.

b) Beam-type CID of PB product m/z 921.7. c) MS3 ion trap CID of m/z 339.3 from (b).

PI 18:0_18:1(9)4.8e5

100 200 300 400 500 600 700 800 900

283.3863.6

419.3 581.4297.0241.0 599.3

281.3cp

s

a)

3.1e5

100 200 300 400 500 600 700 800 900 1000

921.7

283.3 339.4

311.3 419.4 581.3 883.6255.2152.9

cp

s

b)

9.6e4

100 200 300m/z

x 10.0

341.3337.3

339.3281.2

197.1171.1

cp

s

c)

[M-H]-

[M-H+58]-

S-10

Figure S-7: Proposed structure, fatty acyl structural determination, and PB diagnostic ions for

LPI 18:1(9) from direct injection of yeast polar lipid extract. a) Beam-type CID of m/z 597.4. b)

Beam-type CID of PB product m/z 655.4. c) MS3 ion trap CID of m/z 339.3 from (b).

LPI 18:1(9)

100

4.5e4

200 300 400

x 5.0

281.2

339.2197.0171.0

cp

s

8.5e5

100 200 300 400 500 600

521.5

597.4281.3

255.2 535.5153.0 241.0 315.1

423.4

cp

sa)

c)

[M-H]-

m/z

9.4e4

100 200 300 400 500 600 700

655.4

339.3

255.2

283.3

cp

s

b)[M-H+58]-

S-11

Figure S-8: Proposed structure, fatty acyl structural determination, and PB diagnostic ions for PC

16:1(9)_18:1(9) from direct injection of yeast polar lipid extract. a) Beam-type CID of m/z 742.6

([M-15]-). b) Beam-type CID of precursor positive ion m/z 758.6. c) Beam-type CID of PB

product m/z 816.6.

9.6e5

100 200 300 400 500 600 700

281.3

742.6

253.2 478.3506.3

1.0e6

100 200 300 400 500 600 700 800

184.1

758.6

125.0 577.4

4.0e5

100 200 300 400 500 600 700 800

184.1 816.6

798.6125.0

648.4676.5 702.6674.5

657.6cps

cps

cps

a)

b)

c)

PC 16:1(9)_18:1(9)[M-15]-

[M+H]+

m/z

[M+H+58]+

S-12


LPC 16:1(9) from direct injection of yeast polar lipid extract. a) Beam-type CID of m/z 493.3. b)

Beam-type CID of PB product m/z 552.4.

5.3e5

100 200 300 400 500 600

184.1

534.4552.4

420.2394.2 516.4

412.2

438.2104.1

cps

3.7e5

100 200 300 400 500

184.1

476.3

104.1 417.3311.3

493.3

cp

sa)

b)

LPC 16:1(9)

[M+H+58]+

[M+H]+

m/z

S-13


LPE 16:1(9) from direct injection of yeast polar lipid extract. a) Beam-type CID of m/z 450.1. b)


5.9e4

100 150 200 250 300 350

x 5.0311.3

253.2

197.0

293.2171.0cps

6.1e5

100 200 300 400 500

311.3

508.3283.2253.2

340.3

cps

2.5e6

100 200 300 400 500

253.0

450.1283.1

cps

a)

b)

c)

LPE 16:1(9)[M-H]-

[M-H+58]-

m/z

S-14


PS 16:1(9)_18:1(9) from direct injection of yeast polar lipid extract. a) Beam-type CID of m/z

758.7. b) Beam-type CID of PB product m/z 816.6. c) MS3 ion trap CID of m/z 311.3 from (b).

d) MS3 ion trap CID of m/z 339.3 from (b).

7.3e4

100 150 200 250 300 350

x 5.0281.2

340.2339.3197.1

171.1

cp

s

7.1e4

100 150 200 250 300 350

x 5.0 311.2

253.1197.0171.0c

ps

2.2e5

100 200 300 400 500 600 700 800

729.5

816.6

311.4 447.3389.3339.4253.2

281.3

572.2407.2 754.6465.3153.0

cp

s

2.1e5

100 200 300 400 500 600 700 800

389.2671.4

407.2281.3253.2

417.3 758.7153.0 435.3

cp

s

a)

b)

c)

d)

PS 16:1(9)_18:1(9)

[M-H]-

[M-H+58]-

m/z

S-15


LPS 18:1(9) from direct injection of yeast polar lipid extract. a) Beam-type CID of m/z 522.3. b)


2.1e5

100 200 300 400 500 600

435.3153.0

522.3

253.2 281.3

cps

a)

b)

LPS 18:1(9)[M-H]-

[M-H+58]-

2.9e4

100 200 300

x 10.0

341.3337.3

339.3281.2197.1171.1

cps

c)

m/z

1.5e5

100 200 300 400 500 600

580.3297.2

493.4339.3152.9

311.2

522.3377.1

506.3

281.2

cps

S-16


PA 16:0_16:1(9) from direct injection of yeast polar lipid extract. a) Beam-type CID of m/z 645.4.


3.5e5

100 200 300 400 500 600 700

703.5255.2391.3311.3

409.3

447.3153.0

465.3

1.3e5

100 200 300 400 500 600 700

255.2 645.4

391.3409.3

153.0601.6

253.2

cp

scps

4.2e4

100 200 300

253.1

311.1

197.0

171.0 293.2

cps

a)

b)

c)

PA 16:0_16:1(9) [M-H]-

[M-H+58]-

m/z

S-17


LPA 18:1(9) from direct injection of yeast polar lipid extract. a) Beam-type CID of m/z 435.4.


1.2e5

100 200 300

333.3335.3

339.4197.2 281.4171.1

cps

1.8e5

100 200 300 400 500

152.9

493.4

457.4268.2256.2 296.2339.3

cps

b)

c)

LPA 18:1(9)cps

a)1.4e5

100 200 300 400

153.0

435.4

253.3171.1

281.4

10 x[M-H]-

[M-H+58]-

m/z

S-18

Figure S-15: Application of direct infusion ESI-PB-MS/MS on a PC standard, PC 16:0/18:2 (9Z,

12 Z). Beam-type CID of m/z 816.6, the first generation PB product of PC16:0/18:2, leads to the

formation of two pairs of C=C diagnostic ions at m/z 650/676 and 690/716. The structure of

diagnostic ions are shown below the spectrum.

(1) Han, X.; Yang, K.; Gross, R. W. Mass Spectrom. Rev. 2012, 31, 134. (2) Hager, J. W. Rapid Commun. Mass Spectrom. 2002, 16, 512. (3) Collings, B.; Stott, W.; Londry, F. J. Am. Soc. Mass. Spectrom. 2003, 14, 622. (4) Hager, J. W.; Yves Le Blanc, J. C. Rapid Commun. Mass Spectrom. 2003, 17, 1056.

690.5

716.5

650.4

676.5

M = 758.6

150 250 350 450 550 650 750 850

m/z

184.1

816.6

798.5

x 3.0

690.5716.5650.4

676.5

M+58 Da

100R

el. Int.

(%

)

Documents

Electronic Supplementary Information Method of coupling ... · Figure S-1: MS2 CID of a side reaction product at m/z 876 from UV irradiation of solution ... ion PIS m/z 153.1 for