Cobalt carbonyl complexes as probes for alkyne-tagged lipids.

Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a "tag" that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV(349nm). The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity "pull-down" strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection.

fetal bovine serum, 1% penicillin streptomycin, and 0.25 mg/ml fatty acid-and endotoxin-free BSA (Sigma-Aldrich, St. Louis, MO)]. Cells were incubated with 3 ml labeling media per dish for either 20 h (in the case of fatty acid labeling) or 5 h (in the case of glycerophospholipid labeling) in a humidifi ed chamber at 37°C/5% CO 2 . After labeling, each plate of cells was washed twice with 5 ml DPBS (Mediatech, Inc., Manassas, VA), then cells were scraped into 600 µl ice-cold 0.1N HCl:MeOH (1:1) and transferred to microcentrifuge tubes (Laboratory Product Sales, Rochester, NY). Cellular lipids were extracted by the addition of 300 µl ice-cold CHCl 3 to the scraped cells, vortexed for 5 min at 4°C, and then centrifuged at 4°C for 5 min at 18,000 g . The extracted lipids were stored at Ϫ 20°C until analysis.
Alkyne cobalt octacarbonyl complexation and enrichment. Stock solutions of the alkynyl fatty acids or phospholipids were made up in methanol (1 mM). Samples (20 µl of each stock solution) were prepared in microcentrifuge tubes equipped with a stir bar and diluted to 200 µl with methanol. An aliquot (50 µl) of the initial sample was reserved for analysis. To the remaining sample was added Co 2 (CO) 8 (5 mg) followed by stirring at room temperature for 30 min. The solution should be dark orange-brown at this stage of the reaction. 2-Diphenylphosphinoethyl-functionalized silica gel (50 mg) was added to the solution and heated at 55°C with vigorous stirring. After 2 h, the slurry was transferred to a spintube, a microcentrifuge tube fi tted with a removable 0.45 µm nylon fi lter. The reaction tube was washed with methanol (200 µl) and added to the spin-tube. Then the sample was centrifuged at 13,200 g for 5 min. The silica gel was washed with additional methanol for phospholipids or 1% HOAc/methanol for fatty acids (200 µl × 2), vortexed, and centrifuged. This fi ltrate was reserved as the wash sample. The fi lter containing the silica gel was transferred to a new microcentrifuge tube. Methanol (400 µl) and Fe(NO 3 ) 3 (40 mg) was added and stirred at room temperature for 30 min. The sample was centrifuged at 13,200 g for 5 min, then the silica was washed with additional methanol or acidic methanol (200 µl) as described above. This sample is referred to as the release. See Scheme 1 for the process .
Quantitation based on cobalt complex 349 nm UV. The CHCl 3 layer from a Bligh and Dyer cellular extraction was removed and transferred to a new microcentrifuge tube. The sample was concentrated on a SpeedVac and resuspended in methanol (200 µl). To the sample was added 7a -palmitic acid (100 nmol) as an internal standard and Co 2 (CO) 8 (5 mg). After tag, making the alkyne functional group a potential "tag" for tracking lipids in biology as shown in Scheme 1 ( 13 ). We report here that the alkyne cobalt complexes themselves can be used as reporter tags for lipid classes and molecular species, making quantitative analysis of individual molecules or lipid classes straightforward. We also demonstrate that the strategy is not limited to a terminal alkyne, as is the case for "click" cycloaddition, expanding the variety of structures that can be used as surrogate lipids.

Materials
Commercial anhydrous CHCl 3 was used as received. Purifi cation by column chromatography was carried out on silica gel, and TLC plates were visualized with phosphomolybdic acid. 2-Diphenylphosphinoethyl functionalized silica gel was purchased from Aldrich chemicals. Co 2 (CO) 8 was obtained from Strem Chemicals. 1 H, 13 C, and 31 P NMR spectra were collected on a 300 MHz NMR. The syntheses of di-15a -PPC and its corresponding lyso-15a -PC have been previously described ( 13 ). The detailed synthetic procedures for the preparation of the lipids shown in Fig. 1 are presented in the supplementary data.

Alkynyl lipid cellular incorporation and enrichment
Incorporation into RAW cells. RAW 264.7 cells were obtained from the American Type Culture Collection and maintained in Dulbecco's Modifi ed Eagle Medium (DMEM) supplemented with high glucose and pyridoxine hydrochloride (Gibco, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin streptomycin (Gibco, Grand Island, NY). Cells were plated onto 60 mm tissue culture dishes (Corning Inc., Corning, NY) at 2 × 10 6 cells/ dish and incubated in a humidifi ed chamber at 37°C/5% CO 2 for either 3 h (in the case of fatty acid labeling) or 24 h (in the case of glycerophospholipid labeling) to allow cells to adhere to the plates. Plating media and nonadherent cells were removed and replaced with freshly sonicated labeling media [50 µg/ml 7a -16:0 or 15a -16:0, or 2 mg/ml di-15a -PPC in DMEM containing 10% heat-inactivated The formation of the cobalt-alkyne complexes could be readily monitored by HPLC/UV. The peak due to the alkyne disappears after the Co 2 (CO) 8 treatment and a much less polar compound corresponding to the cobaltalkyne complex generally appears in the chromatogram. The cobalt-alkyne complexes have a characteristic absorbance at 349 nm with a molar absorption coeffi cient found to be ‫ف‬ 3,700 M Ϫ 1 cm Ϫ 1 for several of the alkyne-cobalt complexes described here. Thus, monitoring the chromatogram at 349 nm immediately identifi es the complex and permits a quantitative analysis of that species based upon . Some of the alkyne complexes could be isolated and further characterized by NMR analysis. This generally showed a downfi eld shift of acetylenic protons from 2.1 to 6.3 ppm, and a more modest shift of propargylic protons from 2.1 to 2.9 ppm (see supplementary Fig. I).
HPLC/MS proved to be a useful tool for the analysis of complex lipid mixtures containing alkynyl-tagged lipids. The intact alkynyl-cobalt phospholipid complexes produced strong molecular ions on ion-trap mass spectrometers, whereas analysis on quadrupole instruments gave inconsistent results. With trifluoroacetic acid or ammonium acetate additive, a strong [M+H] + peak was observed in the positive mode. Some fragmentation corresponding to sequential CO loss was also detected ( Fig. 2 ). The [M+OAc] Ϫ adduct could also be seen in negative mode. For phospholipid alkynes, the cobalt complexes were less readily detected than the parent alkynes, mass spectral analysis being less sensitive for the bis-dicobalt complex compared with the mono -complexed species which was, in turn, observed at lower signal than the parent phospholipid. Increasing the number of alkynes, and hence the number of cobalt species bound to the molecule, results in lower signal intensity.

Cobalt complex ligation to solid support and release
Phosphines are good ligands for alkyne-cobalt complexes, and phosphines on polystyrene ( 18 ), Novagel ( 20 ), and silica gel were examined as potential solid supports for ligation of the alkynyl lipid cobalt complexes. For small nonpolar alkynes, polystyrene-phosphine beads worked well. The solid-supported phosphine was added to a solution of the cobalt-alkyne complex in methanol and heated at 70°C with gentle stirring under argon for an hour, and under these conditions, immobilization on beads was essentially complete. Polystyrene beads were problematic, however, for studies with polar lipids, and the silica-supported phosphine was ultimately used in most of our studies.
For immobilization of alkyne lipid analogs on the silicasupported phosphine, the reaction could be carried out at 55°C in methanol. This lower temperature eliminated transesterifi cation of the phospholipids, a reaction that was sometimes observed at higher temperatures and evident by the formation of fatty acid methyl esters. In addition, there was signifi cantly less nonspecifi c binding of the phospholipids to the silica support as compared with polystyrene. Capture of fatty acids and phospholipid alkynes was nearly complete on silica-phosphine beads after an the reaction mixture had stirred at room temperature for 30 min, it was analyzed by HPLC with UV detection at 349 nm. The area under curve for the di-15a-PPC-cobalt complex was corrected by a factor of two, taking into account it has two alkynes. For mass spectral quantitation, a sample was equally divided and half subjected to UV analysis of the cobalt complex as described. The other half was spiked with 33:0 plasmanyl PC (33:0 PCe) (20 nmol) as an internal standard and analyzed by LC/MS (see the supplementary data). A response factor was determined using authentic standards of di-15a-PPC and 33:0 PCe. Phospholipids were detected as their M+H adduct. Dimers of the phospholipids were also detected and were included in the integration. For both quantitation methods, the reaction mixture was analyzed on an Ascentis C8 column using a mobile phase consisting of A (5 mM NH 4 OAc in H 2 O) and B (5 mM NH 4 OAc in methanol). The phospholipids were eluted with a gradient of 80 to 100% B over 10 min, held for 15 min, and back to 80% B over 5 min. Mass spectral analysis was carried out on an ion trap in positive ionization mode with the following ESI source parameters: capillary temperature 200°C, capillary voltage 17 V, spray voltage 4.5 kV, tube lens offset 15 V, and sheath gas (nitrogen) 20.
Alkynyl lipid enrichment cells. The CHCl 3 layer from the Bligh and Dyer extraction was removed and transferred to a new microcentrifuge tube. Two samples were combined, concentrated, and resuspended in methanol (200 µl). The enrichment of the di-15a--PPC was carried out as described above. Prior to mass spectral analysis, the excess Fe(NO 3 ) 3 was removed from the sample. The release sample was concentrated, then resuspended in 400 µl CHCl 3 , 200 µl methanol, and 200 µl 0.1 N HCl. The sample was vortexed for 1 min and centrifuged at 13,200 g for 1 min to separate the layers. The CHCl 3 layer containing the phospholipids was removed and concentrated. The sample was resuspended in 200 µl methanol and analyzed by LC/MS using the conditions described above. Detailed procedures for separation and quantitation of lipid classes along with examples of alkynyl lipid enrichment from cell culture are presented in the supplementary data.

Cobalt complex formation and characterization
Conditions suitable for use in enriching alkyne-tagged lipids from cells were sought so that organelle distribution and metabolism studies could be carried out with tagged precursors. In general, cobalt complexes were formed by addition of Co 2 (CO) 8 to a solution of the alkyne in methanol. The cobalt reagent itself is unstable and readily oxidizes upon exposure to air, evident by a color change from orange to purple. In contrast, the cobalt-alkyne complexes are stable as long as they are not exposed to oxidative conditions. Complexes form rapidly at room temperature, generally within 10 min, producing an orange solution. Although methanol is the preferred solvent for lipid substrates, acetonitrile, methylene chloride, isopropanol, and tetrahydrofuran were used in some cases for cobalt complexation. Minimal amounts of water could be used with methanol as a cosolvent, but when it exceeded 20%, signifi cant precipitation of Co 2 (CO) 8 occurred and complexation to the alkyne failed. For assays on alkynyl lipids in cell culture, cells were washed to remove media and scraped into acidic methanol, and the lipids were extracted using a modifi ed Bligh and Dyer protocol ( 21,22 ). the best results. If nonprotic solvents were used in the ferric nitrate oxidation reaction, alkene Z → E isomerization of unsaturated fatty acids and esters attended the release from the solid support, whereas alkene isomerization was not observed in methanol solvent.
Mixtures of alkynyl and natural lipids were made up to test the conditions for separations of the alkynyl-tagged lipids from their natural counterparts. Treatment of the lipid mixtures in methanol with excess Co 2 (CO) 8 at room temperature for 30 min was followed by addition of phosphine on a silica support. This mixture was heated at 55°C for 2 h with vigorous stirring and then transferred to an Eppendorf centrifuge tube fitted with a 0.45 µm nylon fi lter. The solid support was washed and fi ltered several times to remove the nonbound compounds. Subsequent treatment with Fe(NO 3 ) 3 at room temperature for 30 min released the alkyne. This enrichment protocol generally works for both fatty acids and phospholipids. Fig. 3 shows a typical separation of synthetic alkynyl lipids and the corresponding natural compounds. Fig. 3A shows separation of alkynyl analogs of palmitic (a-Palm) hour at 55°C; terminal alkynes appeared to react somewhat faster than their internal alkyne counterparts. Although the number and position of the alkyne in a substrate has some infl uence on the rate of immobilization, all phospholipids studied were successfully captured within 2 h at 55°C. Capture could also be achieved at 37°C for 5 h or overnight at room temperature.
After immobilization on a solid support, beads were washed to remove compounds not bearing the alkyne/cobalt functionality, and the parent alkyne was released from the support by mild oxidation of the cobalt complex. Many oxidants were screened to fi nd conditions that resulted in the highest recovery of the alkyne. Trimethylamine oxide, ceric ammonium nitrate, air, and HCl resulted in the release of 30-50% of alkyne from solid support, whereas KSCN, t BuOOH, and tetrabutyl ammonium fluoride (TBAF) failed in our hands to give measureable free alkyne. Ferric nitrate produced the best results by far ( 14 ), with 70-80% recovery of the alkyne. Upon immobilization of the cobalt-alkyne complex on support, washing with methanol and then release using Fe(NO 3 ) 3 generally provided endogenous phospholipids or fatty acid esters do not generally absorb at this wavelength. This permits quantitative analysis of alkynyl lipids extracted from a biological mixture, e.g., by the use of HPLC/UV 349nm when coupled with addition of a known amount of an alkyne standard that forms a cobalt complex. This method greatly simplifi es the quantitation in that a single standard is used, and response curves need not be generated for every compound of interest. In addition, it greatly simplifi es the detection of alkynyl species in a complex mixture simply by UV analysis at 349 nm.
Model assays. This approach was tested in model assays of alkynyl phospholipids making use of various added alkynyl standards that form cobalt complexes having convenient retention times relative to the phospholipid complexes on normal and reverse-phase HPLC. The mixtures of alkynyl phospholipids and standards were treated with Co 2 (CO) 8 , then analyzed by HPLC with UV detection at 349 nm (see supplementary Fig. II). With addition of an appropriate alkyne standard, either normal-phase or reverse-phase HPLC/UV on the alkyne cobalt complexes can be used to report the amounts of alkynyl lipids present in a mixture. Lipids bearing two alkynyl groups analyze appropriately if the molar absorption is assumed to be twice that of a mono-alkyne. From the results in Table 1 , one can see that the amount of alkyne based on the cobalt quantitation is in good agreement with the theoretical value. and linoleic (a-Lin) acids from the natural acids by cobalt complex formation, "pull down" on the phosphine solid support, wash of natural fatty acids, and release of the alkynyl fatty acids after Fe(NO 3 ) 3 oxidation. The same sequence shown in Fig. 3B was used for treatment of the phospholipid mixture made up of DPPC and three alkynyl analogs of that phosphocholine, di-15a -PPC, di-7a -PPC and P-15a -PPC. The compound 1-palmitoyl 2-acetyl -snglycero-3-phosphocholine [P(OAc)PC] was added as an external standard. DPPC is the dominant phospholipid present in the wash solution, whereas the three alkynyl lipids are all retained on the silica solid support and released upon reaction with ferric nitrate. There is some alkyne in the wash, either from incomplete immobilization onto the solid support or premature oxidation of the cobalt at this step. In addition, there is some crosscontamination evident in the release due to nonspecifi c binding, but the alkynyl lipids are greatly enriched in the cobalt complex catch-and-release strategy. Similar studies were carried out with fatty acids and esters, and similar results were obtained.

Quantitation using cobalt complex
The characteristic absorbance of alkyne cobalt complexes at 349 nm has the potential for use in the detection and quantitation of alkyne lipid surrogates. The extinction coeffi cient for the cobalt complex is essentially the same for terminal and internal alkynes (3,700 M Ϫ 1 cm Ϫ 1 ), and Separation of lipid classes. The lipid extract was treated with Co 2 (CO) 8 and the resultant mixture was analyzed by normal-phase HPLC under conditions in which lipid classes (i.e., phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and PC ) were separated. 15a -P(OAc) PC was added as an internal standard so that the alkynyl phospholipid cobalt complexes could be assayed and lipid class distribution could be determined by the characteristic 349 nm absorption. The chromatographic analysis included the phosphocholines, eluting at ‫ف‬ 36 min (see Fig. 4 ), the ethanolamines (21 min), along with the serines (28 min), inositols (20 min), and free fatty acids (12 min). Triglycerides, diacylglycerols, sterol esters, and other more esoteric phospholipids, such as cardiolipins, were not analyzed in these studies, but the approach illustrated here could be expanded to include a broader range of lipid classes. As shown in Fig. 4 , the alkynyl cobalt-modifi ed phospholipids are distributed almost entirely into the phosphocholine lipid class. Triplicate analysis of lipid extracts from experiments with 7a -palmitic acid enrichment showed that over 96% of the alkynyl phospholipids were present as phosphocholines. For 15a -palmitic acid, some 12-14% of the alkynyl fatty acid was detected in the phosphoethanolamine fraction, the remainder being present as phosphocholines. The total amount of alkynyl phospholipids generated was quantitated based on its UV absorbance relative to the internal standard and is presented in Table 2 (row 1). Incorporation of 15a -palmitic and 7a -palmitic resulted in the formation of 2.4-7.2 and 1.8-4.9 nmol/10 6 cells of alkynyl phospholipids, respectively. There is not a signifi cant difference in the total amount of phospholipid formed for each of the fatty acids, although there is some variation between cell batches. No free fatty acid was detected ( Table 2 , row 3), suggesting that there was signifi cant incorporation into the cell in some esterifi ed form. The alkynyl phospholipid cobalt-complex fractions shown in Fig. 4 were collected and subsequently analyzed by mass spectrometry. The major alkynyl phospholipids identifi ed by m/z of the cobalt complex molecular ion were 15a -PPPC and di-15a -PPC for experiments with 15apalmitic, and the corresponding 7a -PPPC and di-7a -PPC were the major products when 7a -palmitic was the fatty acid added to the cellular media.
Alkynyl lipid affi nity purifi cation. The lipid class separation and subsequent mass spectral analysis provide a preliminary identification of the alkynyl phospholipids. However, minor alkynyl phospholipids may be diffi cult to detect unless specifi cally enriched or captured using the cobalt approach. Analysis of the cobalt pull-downs were carried out on lipid extracts from experiments in which alkynyl fatty acids were incorporated into RAW 264.7 cells.
Alkynyl lipids in cell cultures. The utility of the cobalt complex for quantitative analysis is illustrated for the incorporation of an alkynyl lipid into cells. After incubating RAW 264.7 cells with media enriched with di-15a-PPC for 5 h, the cells were washed to remove excess lipid and scraped into acidic methanol, and the phospholipids were extracted using a modifi ed Bligh and Dyer protocol. The lipid extract was treated with Co 2 (CO) 8 and 7a-palm was added as an internal standard. The mixture was then analyzed by HPLC with UV detection at 349 nm (see supplementary data for a typical chromatogram). The amount of di-15a-PPC that is associated with the cells could then be determined based on its UV absorbance relative to the internal standard. Four separate samples were analyzed by this method, and they were shown to contain 11.9, 7.5, 10.2, and 7.9 nmol/10 6 cells of di-15a-PPC. The fourth sample was also analyzed by mass spectrometry using an unnatural 33:0PCe as a standard. By MS analysis, cells were found to have 7.6 nmol/10 6 cells of di-15a-PPC, a result that is nearly identical to the cobalt quantitative analysis. With these values and the known concentration of lipid used in the incubation, an incorporation of di-15a-PPC in cells of 0.5-0.7% was calculated.
Because the media containing excess di-15a-PPC was washed away, we know that this amount of incorporation does not represent free di-15a-PPC. However, we should point out that it is diffi cult to determine how much of the di-15a -PPC was incorporated into the cell or is merely associated with the outer membrane. From our previous work, we know that some of the di-15a -PPC is indeed incorporated into the cell. In that work, primed RAW 264.7 cells enriched with di-15a -PPC were treated with phorbol 12myristate 13-acetate (PMA) in the presence of n -butanol. The di-15a -PPC was converted to the transphosphatidylation reaction product 32:4 alkynyl PtdBuOH via the phospholipase D metabolic pathway, demonstrating that this metabolic pathway is still active with the alkynyl PC as substrate.

Alkynyl fatty acids in cell culture
RAW 264.7 cells were incubated with 15a-or 7a -palmitic acid for 15 h, then the cells were lysed, and the lipids were extracted. In this experiment, the alkynyl fatty acids should be taken up by the cells and incorporated into a variety of phospholipids and other esters. Subsequent observation of a complex mixture of alkynyl phospholipids indicates that these fatty acids have been successfully used in metabolic processes. Several different analyses, described below, were carried out on these lipid extracts to demonstrate the utility of the cobalt approach in identifying these lipids. Based on that assumption, a careful analysis suggests that a number of minor phospholipid products are present but that the two major products present in the release fractions for both 7a -and 15a -palmitic acid cell incubations are phosphatidyl cholines containing either one or two alkynyl fatty acid esters at sn1 and sn2 . These two lipids account for 59 and 68% of the total phospholipid for 15a -and 7a -palmitate incorporation. This is consistent with the results described above for the mass spectral identifi cation of the cobalt-alkyne complexes.
Other identifi able lipids were found in the release fractions for the 15a -and 7a -palmitate cellular incorporation experiments. In addition to the major phosphocholine class compounds, some plasmalogen and ethanolamine structures are also suggested for some of the minor constituents, all of which had MS characteristics consistent with the presence of one alkynyl palmitoyl group as a glyceryl ester.
Typical chromatograms for these analyses are presented in Fig. 5 . The phospholipids 15a -P(OAc)PC and P(OAc)PC were added to the cell extract to monitor the effi cacy of the pull-down and release strategy. This not only gives us an indication of how well the enrichment is working on a sample by sample basis but also allows us to carry out quantitation of individual phospholipid species. As shown in the Fig. 5 , all of the P(OAc)PC is seen in the wash fractions whereas most of 15a -P(OAc)PC is observed in the ferric nitrate release fraction, indicating that the captureand-release technique is working and that we should have only alkynes enriched in the release sample. This greatly simplifi es the mass spectral analysis and identifi cation of the alkynyl phospholipids.
Because the release fraction in Fig. 5 is the result of an alkyne capture-and-release strategy, one can reasonably assume that most, if not all, of the constituents observed in the HPLC/MS chromatogram have alkyne functionality. Extracts were spiked with 15a -P(OAc)PC, treated with Co 2 (CO) 8 , and analyzed by normal phase HPLC (hexanes/iPrOH/NH 4 OAc gradient) with UV detection at 349 nm.  7a -palmitate, respectively. The slightly higher incorporation of 15a -palmitate is consistent with the slightly higher levels of alkynyl phospholipids present.
HPLC/MS analysis was also carried out on the fatty acid mixture obtained by base hydrolysis of the cellular extract, see Fig. 6 . In addition to 15a-palmitic, this analysis shows that 17a-stearic acid, another alkynyl fatty acid, was present in the mixture. Independent synthesis, extracted ion chromatography, and cobalt complex formation of the two-carbon chain extension product of the alkynyl palmitate confi rms its presence in the lipid extract. For 15a-palmitic acid cellular incubations, 16% of the fatty acid extract is in the chain elongation form, whereas experiments with 7a-palmitic acid give rise to 4% of the metabolic product in the mixture. The terminal alkyne seems to be incorporated somewhat better in the cell and phospholipids than the internal alkyne, and it appears to undergo metabolic chain extension more readily as well. Nonetheless, both compounds are readily incorporated into the lipids of RAW 264.7 cells, and both undergo at least some of normal metabolism expected of endogenous fatty acids.

Conclusions
Tracking the cellular incorporation of lipids is essential to understanding lipid metabolic and signaling processes. Most of the studies to date have utilized radioactive-labeled compounds to monitor changes in lipids during cellular The amount of alkynyl phospholipids was determined relative to the standard for two samples and is presented in Table 2 (row 2). The amount of phospholipids formed from the 15a -palmitic acid is just slightly higher than for the 7a -palmitic acid-labeled cells. In addition, the amount of phospholipid formed based on mass spectral quantitation is in good agreement with the cobalt quantitation of the lipid classes described above. Not only does this result validate the cobalt approach but it also indicates that there is good recovery of the phospholipids after enrichment.
Base hydrolysis and fatty acid analysis. The lipid extract from alkynyl fatty acid incorporation into RAW 264.7 was treated with 3M LiOH to hydrolyze all fatty esters, and fatty acids were then extracted. As the original cell extract contained little, if any, detectable free fatty acids, this extract included all fatty acids that were originally present as esters in the cell. The fatty acid extract was spiked with an alkyne internal standard and treated with Co 2 (CO) 8. Reaction of the fatty acid mixture with Co 2 (CO) 8 followed by HPLC/UV 349nm with an internal standard permitted estimation of total amount of fatty acid incorporation into the cells during the incubation period. By this analysis, we estimate that the total amount of 15a -and 7a -palmitic acid incorporated was 14.4 and 9.0 nmol/million cells, respectively, during the 15 h incubation of the fatty acids with RAW 264.7 cells ( Table 2 , row 4). This corresponds to 12 and 8% total incorporation for 15a -and compounds. Modifi cation of the alkynyl lipid through click chemistry would allow one to identify the alkynyl lipid in a complex mixture. However, quantifi cation of dozens of lipid species simultaneously would not be practical, as standards for these heavily derivatized phospholipids are not commercially available. Finally, conventional click chemistry is only viable for terminal alkynes and results in the permanent modifi cation of the phospholipid of interest.
This work demonstrates that alkynyl probes have the potential to provide a broad-scope method for quantitative analysis of cellular lipid transformations when coupled with an approach involving cobalt complex formation, pull-down on a phosphine solid support, and oxidative release. The utility of these probes is enhanced by powerful chromatographic methods for separation of lipid classes and molecular species, as well as by the fact that the alkynecobalt complex has a signature UV absorption at 349 nm. As shown in these studies, detection of the alkyne lipid analogs has been optimized such that they can be used to processes. Although these procedures are widely used and easily performed, they provide a limited amount of information about the lipid species involved in signaling events. Furthermore, even advanced methods of mass spectrometry have limitations with regard to discriminating very complex mixtures of isobaric species. The main drawback for all of these methods is the inability to discriminate between labeled and naturally occurring lipids in very complex mixtures that contain in excess of 1,000 species of phospholipids.
The use of alkyne tags for substrates has become popular with the interest in click chemistry. Alkyne-modifi ed substrates, in conjunction with click chemistry, offer an approach to capture the substrate through selective modification of the alkyne moiety. While this approach has been successfully used in a wide variety of applications, it has some potential drawbacks as a technique for tracking the cellular incorporation of lipids. The alkyne tag alone is not suffi cient for identifying the modifi ed lipid because the alkynyl lipid is isobaric with naturally occurring