The detergent-solubilized and gel filtration purified rhodopsin was partitioned against

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1 Supplement Jastrzebska et al. Materials and Methods The detergent-solubilized and gel filtration purified rhodopsin was partitioned against H 2 O/MeOH/CHCl 3, and the bottom layer was removed, dried down, and then redissolved in CHCl 3. The CHCl 3 solution was fractionated into 3 different classes of lipids (neutral lipid, glycolipid, and polar lipid) by silicic acid column chromatography subsequently using CHCl 3, acetone, and MeOH (ref 1). The MeOH fraction was dried down and re-dissolved in 10% H 2 O/ 80% MeOH/10%CHCl 3 for mass spectrometry analysis. For detection of glycerophosphatidylcholine (PC) and glycerophosphatidylserine (PS) in the positive mode and nitrogen containing glycerophosphatidylethanolamine (PE) and PS in the negative mode, 0.2% acetic acid and 0.05% piperidine (ref 2) were included, respectively. Electrospray mass spectrometry analysis of glycerophospholipids was performed on a PE/SCIEX API QSTAR PULSAR i Quadruple Time-of-Flight with a nebulization-assisted homemade nanospray ionization source, and the samples were introduced at a flow rate of 0.4 μl/m with a Harvard syringe pump. The homemade nanospray source was constructed as follows. The sample (in a 50 μl Hamilton syringe) was infused into a fused silica capillary tube (i.d x o.d. 360 μ) that was connected to a zero dead volume stainless union. The union was then connected to a 30-cm emitter (tip i.d 30 μ, i.d. 50 x o.d. 360μ; New Objectives, Woburn, MA), and 5.8 kv and -4.5 kv were applied to the union for the positive mode and negative mode, respectively. For a complex mixture of 1

2 glycerophospholipids, the precursor ion experiment should be carried out first to identify the different classes of glycerophospholipids, and then the tandem mass spectrometry (MS/MS) would be followed to analyze the structures of glycerophospholipids. Due to the production of the characteristic polar head group ion of different glycerophospholipids by collision activated dissociation (CAD) mass spectrometry, the following diagnostic mass ions (m/z) were used for identification of each individual class of glycerophospholipids in the precursor ion experiment (ref 3) and listed in Table 1: [CH 2 C(OH)CH 2 HPO 4 ] - ( ) for general glycerophospholipids and glycerophosphatidic acid (ref 4), [H 2 PO 4 CH 2 CH 2 (NCH 3 ) 4 ] + ( ) for PC (ref 5), [CH 2 C(OH)CH 2 PO 4 CH 2 CH 2 NH 2 ] - ( ) for PE (ref 6), [HPO 4 CH 2 CH(OH)CH 2 OH] - ( ) for glycerophosphatidylglycerol (PG) (ref 7), [HPO 4 CH 2 CH(OH)CH 2 OH] - ( ) for glycerophosphatidylinositol (PI) (ref 8), and [NH 3 C(CH 2 )COOH] + (88.040) for PS (GFJ s data). Results: 2

3 Strategies for analysis of glycerophospholipids associated with detergent-solubilized and gel filtration-purified rhodopsin-the gel filtration purified rhodopsin was partitioned against H 2 O/MeOH/CHCl 3, and then the bottom layer was fractionated by silicic acid column chromatography. General fractionation of lipids into neutral lipid, glycolipid, and polar lipid can be accomplished by silicic acid column chromatography subsequently using CHCl 3, acetone, and MeOH (ref 1). The polar lipid containing (MeOH) fraction was first analyzed for glycerophospholipid classes associated with rhodopsin by the precursor ion scanning (PIS) experiment of mass spectrometry in the positive (PC and PS) and the negative (PA, PE, PG, and PI) mode. Due to the unique structural feature of the polar head group of the individual glycerophospholipid class, the head group will be fragmented into the characteristic low molecular ion species by CAD quadruple-time-offlight mass spectrometry (Q-TOF). These characteristic low molecular ion species (ref 2-8) can be utilized for identification of different classes of glycerophospholipids and were shown in Table 1. Then the individual glycerophospholipid molecular identity will be determined by the tandem mass spectrometry (MSMS) experiment in the positive and negative mode that would provide abundant information regarding the fatty-acyl compositions esterified to the glycerophospholipid backbone and the polar head group of the glycerophospholipid. Precursor ion scanning mass spectrometry revealed that PC, PE, and PI were associated with rhodopsin-in order to investigate if different CMC values of detergents would result in different classes of glycerophospholipids associated with detergentsolubilized and gel filtration purified rhodopsin, DM (CMC:0.17 mm), TDM (0.01) and HDM (0.0006) were selected for the studies. Figure 1A, 1B, and 1C showed similar PC 3

4 class ion patterns obtained by PIS of m/z (positive mode) from DM-, TDM- and HDM-solubilized and gel filtration purified rhodopsin samples, respectively. Figure 2A, 2B, and 2C showed similar PE class ion profiles obtained by PIS of m/z (negative mode) from DM-, TDM-, and HDM-solubilized and gel filtration purified samples, respectively. Figure 3A, 3B, and 3C also showed similar PI class ion profiles obtained by PIS of m/z (negative mode) from DM-, TDM-, and HDMsolubilized and gel-filtration-purified samples, respectively. No PG class and PS class ions were observed by PIS of m/z and m/z , respectively (data not shown). It was also very likely that PA was not associated with rhodopsin since no PA class ion profile with less than m/z 730 ([M-H] - ) (most of PA have MW less than MW 730) by PIS of m/z was observed as well (data not shown). Tandem mass spectrometry of [M + H] + ions in the positive mode for PC-The molecular identities of the respective glycerophospholipid class were characterized by either the positive mode (PC) or the negative mode (PE and PI) MSMS. Figure 4A and 4B showed the product ion spectrum of m/z (calculated [M + H] + : ) obtained from PIS of PC class in Figure 1 and indicated that it is 1-stearoyl-2-docosahexaenoyl-snglycero-3-phosphocholine. In addition to the second abundant molecular ion, [M + H] + (m/z ), shown in Figure 4A, the most prominent ion at m/z (calculated m/z ) confirmed that it was a PC molecule (Table 1) and represented a protonated phosphocholine (ref 5). Although the rest of the ion peaks between m/z and appeared minor in Figure 4A, very rich information regarding the fatty-acyl chains at sn-1 and sn-2 still could be obtained upon close examination of the spectrum (Figure 4B). The m/z ([M -R 2 CH=C=O + H] +, calculated m/z: ) and 4

5 ([M -R 1 CH=C=O + H] +, calculated m/z: ) represented the losses of the fatty acyl at sn-2 and sn-1 as a ketene molecule, respectively, where R 2 = R 2 CH 2 and R 1 = R 1 CH 2. It has been reported that the loss of the fatty acyl at sn-2 or sn-1 as a ketene molecule involves the participation of the respective fatty acyl α-hydrogen, and the intensity of [M -R 2 CH=C=O + H] + is greater than [M -R 1 CH=C=O + H] + since the α- hydrogen of the fatty acyl at sn-2 is more labile (ref 5). Therefore, it would be clear to assign docosahexaenoic acid and stearic acid at sn-2 and sn-1, respectively. The losses of the fatty acyl at sn-2 and sn-1 as a free fatty acid molecule has also demonstrated that there is no preferential loss between both positions since the α-hydrogen of the fatty acyl at sn-2 or at sn-1 does not involve the cleavage of the fatty acid from the glycerophosphocholine backbone (ref 5). It is the H + ion that protonates the molecule and involves the cleavage pathway (ref 5). Ions at m/z ([M -R 2 COOH + H] +, calculated m/z: ] and ([M -R 1 COOH + H] +, calculated m/z: ] represented eliminations of free acid moieties from sn-2 and sn-1, respectively, with equal intensities. Two other ions at m/z ([M -C 5 H 14 NO 4 P + H] +, calculated m/z: ) and ([M -N(CH 3 ) 3 + H] +, calculated m/z: ) were assigned for the losses of the phosphocholine head group and N(CH 3 ) 3, respectively. Table 2 summarized the assignments of major molecular identities of PC class obtained in Figure 1 by MSMS of [M + H] + ions in the positive mode. Tandem mass spectrometry of [M-H] - ions in the negative mode for PE and PI-The major molecular identities of PE and PI class ions analyzed in Figure 2 and 3 by negative PIS were further characterized by the negative mode MSMS. Figure 5 showed the product ion spectrum of the deprotonated molecular anion of 1-stearoyl-2-docosahexaenoyl-sn- 5

6 glycero-3-phosphoethanolamine at m/z ([M - H] -, calculated m/z ) shown in Figure 2. Besides the [M - H] - molecular ion and the ion at m/z ([M - R 2 COOH - R 1 CH=C=O - H] -, calculated m/z ) that accounted for the PE polar head group, three major pairs of ions could be also assigned. The most prominent ions pair at m/z ([R 2 COO] -, calculated m/z ) and m/z ([R 1 COO] -, calculated m/z ) represented the fatty carboxylic acid anions at sn-2 and sn-1, respectively. The assignment was based on the previous observation that the pathway leading to the formation of [R 2 COO] - (atty acyl at sn-2) is likely to be more sterically favorable and results in more intense [R 2 COO] - than [R 1 COO] - (ref. 6). The next intense ions pair at m/z ([M - R 2 CH=C=O - H] -, calculated m/z ) and m/z ([M - R 1 CH=C=O - H] -, calculated m/z ) were derived from the losses of the fatty acyl at sn-2 and sn-1 as a ketene molecule, respectively. It has been reported that the loss of the fatty acyl at sn-2 or sn-1 as a ketene molecule is a charge-driven fragmentation process that sterically favors the ketene loss at sn-2 (ref. 6) and results in the greater intensity of [M - R 2 CH=C=O - H] - than that of [M - R 1 CH=C=O - H] -. The third ions pair at m/z ([M - R 2 COOH - H] -, calculated m/z ) and m/z ([M R 1 COOH - H] -, calculated m/z ) accounted for the losses of the fatty acyl at sn-2 and sn-1 as a free fatty acid molecule, respectively. It has been demonstrated that the pathway leading to the neutral loss of free fatty acid from sn-2 and sn-1 is a charge-remote fragmentation process that involves the participation of hydrogen at C-1/C-3 (4 H s) and C-2 (1 H) to produce [M - R 2 COOH - H] - and [M R 1 COOH - H] -, respectively, and result in a higher intensity of [M - R 2 COOH - H] - than that of [M R 1 COOH - H] - (ref 6). Based on the above discussion, docosahexaenoic and stearic acid 6

7 were assigned to be esterified at sn-2 and sn-1, respectively. It is also worth noting that the intensity of [M - R 2 CH=C=O - H] - / [M R 1 CH=C=O - H] - pair was higher than that of the corresponding [M - R 2 COOH - H] - /[M R 1 COOH - H] - pair due to the basic nature of the [M - H] - precursor ion of the PE class in the gas phase that underwent the preferential loss as a ketene over the loss as an acid. (ref 6). Table 3 summarized the assignments of major molecular identities of PE class shown in Figure 2 by MSMS of [M - H] - ions in the negative mode. As to the last PI class, Figure 6 showed the product ion spectrum of the deprotonated molecular anion of 1-palmitoyl-2-arachidonoyl-sn-glycero-3- phosphoinositol at m/z ([M - H] -, calculated m/z ). Besides the [M - H] - molecular ion and the ion at m/z [C 6 H 10 O 8 P] - or ([M - R 2 COOH - R 1 CH=C=O C 3 H 6 O 2 - H] -, calculated m/z ) that accounted for the PI polar head group, three major pairs of ions could be also observed as in the case for PE. The assignment of arachidonate and palmitate esterified at sn-2 and sn-1, respectively, was based on the previous observation that the loss of the fatty acyl group as a free fatty acid or ketene group is a charge-driven fragmentation process and occurs more favorably at sn-2 than at sn-1 (ref 8). Therefore, the intensity of m/z ([M - R 2 CH=C=O - H] -, calculated m/z ] and m/z ([M - R 2 COOH - H] -, calculated m/z ) pair was greater than that of the corresponding m/z ([M R 1 CH=C=O - H] -, calculated m/z ] and m/z ([M R 1 COOH - H] -, calculated m/z ) pair. However, due to the acidic nature of the precursor ion [M - H] - of PI class in the gas phase, the loss of the fatty acyl group as a free fatty acid is more favorable than the loss as a ketene (ref 8), so the intensity of the pair of [M - R 2 COOH - H] - and [M R 1 COOH - 7

8 H] - was greater than that of the corresponding pair of [M - R 2 CH=C=O - H] - and [M R 1 CH=C=O - H] - as observed in Figure 6. It has been reported that the ion abundance ratio of [R 2 COO] - / [R 1 COO] - is collision energy-dependent (ref 8). The higher collision energy resulted in more favorable productions of [M - R 2 COOH - H] - (m/z ) and [M - R 2 COOH - C 6 H 10 O 5 - H] - (m/z , calculated m/z ) followed by further dissociation to form [R 1 COO] - that consequently surpassed [R 2 COO] - deriving from [M R 1 COOH - H] - (m/z ) and [M R 1 COOH - C 6 H 10 O 5 - H] - (m/z , calculated m/z ) that were less produced. Therefore, it would be better to assign fatty acyl groups at sn-2 and sn-1 for PI based on the ion abundances between the pair of [M - R 2 COOH - H] - and [M R 1 COOH - H] -. Table 3 summarized the assignments of major molecular identities of the PI class shown in Figure 3 by MSMS of [M - H] - ions in the negative mode. Table 1 8

9 Characteristic low molecular ions generated from different glycerophospholipids polar head groups upon CID. Polar head group m/z Corresponding Characteristic ion species PA [CH 2 C(OH)CH 2 HPO 4 ] -a PC [H 2 PO 4 CH 2 CH 2 (NCH 3 ) 4 ] + PE [CH 2 C(OH)CH 2 PO 4 CH 2 CH 2 NH 2 ] - PG [HPO 4 CH 2 CH(OH)CH 2 OH] - PI [C 6 H 10 O 8 P] - PS b [NH 3 C(CH 2 )COOH] + a It is also a common ion that could be generated from the glycerolphospholipid backbone b GFJ s unpublished results Table 2 9

10 Summary of assignments of major molecular identities of PC class obtained in Figure 1 by the positive mode MSMS. m/z Calculated Fatty acid in sn-1 Fatty acid in sn-2 [M + H]+ R 1 COOH R 2 COOH C 40 H 81 NO 8 P Palmitic (MW: ) (C16:0) C 16 H 32 O 2 CH 3 (CH 2 ) 14 COOH R 1 : C 1 5 H 31 ( ) Palmitic R 2 : C 1 5 H 31 ( ) C 42 H 83 NO 8 P Palmitic Oleic ( ) R 1 : C 1 5 H 31 ( ) (C18:1, 9-cis) C 44 H 81 NO 8 P C 44 H 87 NO 8 P Linoleic ( ) (C18:2,9,12-di-cis) C 18 H 32 O 2 CH 3 (CH 2 ) 4 CH=CH CH 2 CH=CH(CH 2 ) 7 COOH R 1 : C 1 7 H 31 ( ) Stearic ( ) (C18:0) C 18 H 36 O 2 CH 3 (CH 2 ) 16 COOH R 1 : C 1 7 H 35 ( ) C 18 H 34 O 2 CH 3 (CH 2 ) 7 CH=CH(CH 2 ) 7 COOH R 2 : C 1 7 H 33 ( ) Linoleic R 2 : C 1 7 H 31 ( ) Oleic R 2 : C 1 7 H 33 ( ) C 46 H 81 NO 8 P Palmitic Docosahexaenoic ( ) R 1 : C 1 5 H 31 ( ) (C22:6, 4, 7, 10, 13, 16, 19-all-cis) C 22 H 32 O 2 CH 3 CH 2 CH=CHCH 2 CH=CHCH 2 CH=CHCH 2 CH=CH CH 2 CH=CHCH 2 CH=CH(CH 2 ) 2 COOH R 2 : C 21 H 31 ( ) 10

11 C 46 H 85 NO 8 P Stearic R 1 : C 1 7 H 35 ( ) Arachidonic ( ) (C20:4, 5, 8, 11, 14-all-cis) C 48 H 85 NO 8 P Table 3 Stearic R 1 : C 1 7 H 35 ( ) C 20 H 32 O 2 CH 3 (CH 2 ) 4 CH=CHCH 2 CH=CHCH 2 CH=CHCH 2 CH=CH (CH 2 ) 3 COOH R 2 : C 1 9 H 31 ( ) Docosahexaenoic R 2 : C 21 H 31 ( ) Summary of assignments of major molecular identities of the PE class obtained in Figure 2 by the negative mode MSMS. m/z Calculated Fatty acid in sn-1 [M - H] - R 1 COOH Fatty acid in sn-2 R 2 COOH C 43 H 73 NO 8 P C 43 H 75 NO 8 P Palmitic (MW: ) Arachidonic ( ) R 1 : C 1 5 H 31 ( ) R 2 : C 1 9 H 31 ( ) Palmitic Docosapentaenoic ( ) R 1 : C 1 5 H 31 ( ) (C22:5, 4, 7, 10, 13, 16-all-cis) C22H34O2 CH3CH2CH2CH2CH2-CH=CH-CH2-CH=CH-CH2- CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)2COOH R 2 : C 21 H 33 ( ) C 45 H 77 NO 8 P Stearic ( ) Docosahexaenoic ( ) R 1 : C 1 7 H 35 ( ) R 2 : C 21 H 31 ( ) C 45 H 79 NO 8 P Stearic R 1 : C 1 7 H 35 ( ) Docosapentaenoic R 2 : C 21 H 33 ( ) 11

12 Table 4 Summary of assignments of major molecular identities of the PI class obtained in Figure 3 by the negative mode MSMS. m/z Calculated Fatty acid in sn-1 Fatty acid in sn-2 [M - H] C 45 H 78 O 13 P Palmitic (MW: ) Arachidonic ( ) R 1 : C 1 5 H 31 ( ) R 2 : C 1 9 H 31 ( ) C 47 H 82 O 13 P C 49 H 82 O 13 P Stearic ( ) Arachidonic ( ) R 1 : C 1 7 H 35 ( ) R 2 : C 1 9 H 31 ( ) Stearic ( ) Docosahexaenoic ( ) R 1 : C 1 7 H 35 ( ) R 2 : C 21 H 31 ( ) 12

13 Figure Legends Figure 1. PC class ion patterns obtained by PIS of m/z (positive mode) from (A) DM-, (B) TDM- and (C) HDM-solubilized and gel filtration purified rhodopsin samples, respectively. Figure 2. PE class ion profiles obtained by PIS of m/z (negative mode) from (A) DM-, (B) TDM- and (C) HDM-solubilized and gel filtration purified rhodopsin samples, respectively. Figure 3. PI class ion profiles obtained by PIS of m/z (negative mode) from (A) DM-, (B) TDM- and (C) HDM-solubilized and gel filtration purified rhodopsin samples, respectively. Figure 4. (A) The product ion spectrum of the singly charged precursor ion of m/z (1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, [M + H] +, calculated m/z ) derived from PIS of the PC class in Figure 1. The PC polar head group at m/z ([H 2 PO 4 CH 2 CH 2 (NCH 3 ) 4 ] +, calculated m/z ) is always the most prominent ion in the positive mode MSMS of PC class. (B) Informative data could also be obtained by scaling up the same spectrum between m/z 500 and m/z 820 showing the neutral losses of the fatty acyl ([M -R 2 COOH + H] + at sn-2 and [M -R 1 COOH + H] + 13

14 at sn-1) as free acid molecules and the neutral losses of the fatty acyl ([M -R 2 CH=C=O + H] + at sn-2 and [M -R 2 CH=C=O + H] + at sn-1) as ketene molecules where R 2 = R 2 CH 2 and R 1 = R 1 CH 2. Figure 5. The product ion spectrum of the singly charged precursor ion of m/z (1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, [M - H] -, calculated m/z ) obtained from PIS of PE class in Figure 2. Besides the [M - H] - molecular ion and the ion at m/z ([M - R 2 COOH - R 1 CH=C=O - H] -, calculated m/z ) accounted for the PE polar head group, three major pairs of ions could be also observed and provided enough information for molecular identity characterization. Figure 6. The product ion spectrum of the singly charged precursor ion of m/z (1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol, [M - H] -, calculated m/z ) obtained from PIS of PI class in Figure 3. In addition to the [M - H] - molecular ion and the ion at m/z ([C 6 H 10 O 8 P] - or [M - R 2 COOH - R 1 CH=C=O C 3 H 6 O 2 - H] -, calculated m/z ) accounted for the PI polar head group, three major pairs of ions could be also observed and sufficed information for structural identification. 14

15 References 1. White DC, Ringleberg DB. Signature Lipid Biomarker Analysis. In: Burlage RS, Atlas R, Stahl D, Geesey G, Sayler (Eds), Techniques in Microbial Ecology. Oxford University Press, New York, NY, pp Lytle CA, Gan YD, White DC. Electrospray Ionization/Mass spectrometry Compatible Reversed-Phase Separation of Phospholipids: Piperidine as a Post Column Modifier for Negative Ion Detection. J. Microbiol. Methods 2000;41: Pulfer M, Murphy RC. Electrospray Mass Spectrometry of Phospholipids. Mass Spectrometry Reviews. 2003;22: Hsu FF, Turk J. Charg-Driven Fragmentation Processes in Diacyl Glycerophosphatidic Acids Upon Low-Energy Collisional Activation. A Mechanistic Proposal. J. Am. Soc Mass Spectrom. 2000;11: Hsu FF, Turk J. Eletrospray Ionization/Tandem Quadrupole Mass Spectrometric Studies on Phosphatidylcholines: The Fragmentaion Processess. J. Am. Soc Mass Spectrom. 2003;14:

16 6. Hsu FF, Turk J. Charg-Remote and Charg-Driven Fragmentation Processes in Diacyl Glycerophophoethanolamine upon Low-Energy Collisional Activation. A Mechanistic Proposal. J. Am. Soc Mass Spectrom. 2000;11: Hsu FF, Turk J. Studies on Phosphatidylglycerol with Triple Quadrupole Tadem Mass Spectrometry with Electrospray Ionization: Fragmentation Processes and Structural Characterization. J. Am. Soc Mass Spectrom. 2000;11: Hsu FF, Turk J. Characterization of Phosphatidylinositol, Phosphatidylinositol-4- phosphate, and Phosphatidylinositol-4,5-bisphosphate by Eletrospray Ionization Tandem Mass Spectrometry: A Mechanistic Study. J. Am. Soc Mass Spectrom. 2000;11:

17 T O F P r e C u r s o r 0 M C A s c a n s f r o m S a m p l e 3 ( 1 ' '. H D M R h o i n u l 1 0 % C H C l % H 2 O 8 0 %... M a x e 4 c o u n t s e e Fig 1A HDM m / z, a m u C H C l 3 T O F P r e C u r s o r 0 M C A s c a n s f r o m S a m p l e 2 ( 1 '. T D M R h o i n u l 1 0 % 1 0 % H 2 O 8 0 %... M a x c o u n t s Fig 1B T O F P r e C u r s o r 0 M C A s c a n s f r o m S a m p l e 2 ( 1 '. D M R h o i n u l 1 0 % C H m C / lz 3, 1a 0 m % u H 2 O 8 0 % M... M a x c o u n t s TDM DDM Fig 1C m / z, a m u

18 T O F P r e C u r s o r 0 M C A s c a n s f r o m S a m p l e 2 ( 1 '. H D M R h o i n u l 1 0 % C H C l % H 2 O 8 0 % M... M a x c o u n t s Fig 2A HDM m / z, a m u T O F P r e C u r s o r 0 M C A s c a n s f r o m S a m p l e 2 ( 1 '. T D M R h o % H 2 O 1 0 % C H C l % M e O H M a x c o u n t s Fig 2B TDM T O F P r e C u r s o r 0 M C A s c a n s f r o m S a m p l e 2 ( 1. D M R h o i n u l 1 0 % H 2 O m / 1z 0, % a m Cu H C l % M e O... M a x c o u n t s Fig 2C DDM m / z, a m u

19 T O F P r e C u r s o r 0 M C A s c a n s f r o m S a m p l e 5 ( 3 '. H D M R h o i n u l 1 0 % C H C l % H 2 O 8 0 % M e... M a x c o u n t s Fig 3A HDM T O F P r e C u r s o r 0 M C A s c a n s f r o m S a m p l e 5 ( 3 '. T D M R h o % H 2 O 1 0 % m C H/ z C, al 3 m 8u 0 % M e O M a x c o u n t s Fig 3B TDM T O F P r e C u r s o r 0 M C A s c a n s f r o m S a m p l e 5 ( 3 '. D M R h o i n u l 1 0 % H 2 m O / z 1, 0 a% m Cu H C l % M e O... M a x c o u n t s Fig 3C DDM m / z, a m u

20 +TO F Product (834.6): 31 M C A scans from Sam ple 9 (6. 2nd LO AD M eo H FR 0.9x conc. in 0.2%... a= e-004, t0= e e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e [H 2 PO 4 CH 2 CH 2 (NCH 3 ) 4 ] M ax. 3.7e4 counts. Fig 4A [M + H] m /z, a m u +TO F Product (834.6): 31 M CA scans from Sam ple 9 (6. 2nd LO AD M eo H FR 0.9x conc. in 0.2%... a= e-004, t0= e+001 M ax. 3.7e4 counts Fig 4B [M - R 2 COOH + H] + [M - C 5 H 14 NO 4 P + H] [M - R 2 CH=C=O + H] + [M - R 1 COOH + H] [M - N(CH 3 ) 3 + H] [M - R 1 CH=C=O + H] m /z, a m u

21 -TOF Product (790.6): 16 MCA scans from Sample 33 (28. MeOH Fr of LOAD solution in 500 ul a= e-004, t0= e+001 [R 2 COO] [R 1 COO] Max Fig [M - R 2 CH=C=O - H] [M - R 2 COOH - R 1 CH=C=O - H] [M [M - R 2 COOH - H] [M - R COOH - H] m/z, amu 5 [M - R 1 CH=C=O - H]

22 -TOF Product (885.6): 22 MCA scans from Sample 26 (21. MeOH Fr of LOAD solution in 500 ul a= e-004, t0= e+001 [R 1 COO] Max counts. Fig [R 2 COO] [C 6 H 10 O 8 P] [M - R 2 COOH-C 6 H 10 O 5 -H] - [M - R 1 COOH-C 6 H 10 O 5 -H] - [M - H] [M R [M - R 2 CH=C=O - H] - 2 COOH - H] [M R CH=C=O - H] [M - R 1 COOH - H] m/z, amu