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SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards

互联网2013-11-13

1082

实验步骤

Generating labeled coa standards

(1) Thaw frozen cells directly into the SILEC expansion medium.

(2) For Hepa 1c1c7 (adherent) cells, allow cells to reach confluence (3–4 d), wash two times with PBS, trypsinize and split 1:5 into SILEC expansion medium. For S2 (suspension) cells, when cells have reached ~1 × 10⁷ cells per ml, split into a new flask containing SILEC expansion medium at a concentration of 2 × 10⁶ cells.

(3) Repeat Step 2, passaging cells in SILEC expansion medium three more times. At this point, CoA species should be greater than 98% labeled. If cell growth or morphological characteristics are considerably affected, see TROUBLESHOOTING.

NOTE: pause poInt Harvest and freeze down one plate of labeled cells in SILEC expansion medium containing 10% (vol/vol) DMSO. These cells can be thawed to seed future SILEC expansions.

(4) After the fourth passage, allow cells to reach confluence or 1 × 10⁷ cells per ml. Proceed to Step 6 if you do not want the ultimate incorporation of stable isotope label. Alternatively, perform Step 5.

(5)(Optional) Wash cells with 10 ml of PBS, replace with SILEC ultra-labeling medium and incubate for 24 h. Perform this step if you want the highest level of stable isotope labeling, and then continue to Step 6. To produce purified [¹³ C₃ ¹⁵ N]-CoASH, continue to Step 27.

testing of sIlec labeling

(6) Short-chain acyl-CoA acid extraction (Steps 6–9). Harvest one plate of cells by scraping gently into the medium and briefly spin down at 1,000g. The remaining plates should be kept until the first plate has been tested (Steps 7–20).

(7)Resuspend cells in 1 ml of ice-cold extraction solution (10% (wt/vol) TCA).

(8)Sonicate cells for 30 s on ice at a rate of 1 pulse per s.

(9) Spin the sonicated cell lysate at 15,000g for 5 min at 4 °C to precipitate the protein pellet.

(10)SPE (Steps 10–16). Using a vacuum manifold, condition Oasis HLB SPE columns with 1 ml of methanol.

(11)Equilibrate columns with 1 ml of water.

(12) Load acid-extracted lysate (supernatant) onto the columns.

(13)Wash the columns with 1 ml of water.

(14)Elute CoA compounds into fresh tubes by applying three subsequent applications of 0.5 ml of elution solution.

(15) Dry the samples under nitrogen.

(16) Resuspend in 50 µl of 5% (wt/vol) SSA.

(17)LC-MS analysis (Steps 17–20). Using the LC-MS specifications in Figure 2, perform a CID neutral loss scan of 507 amu,scanning a parent mass range of m/z = 750–1,200 amu. Sample injections of 10 µl should be used for analysis.

(18) Characterizing the CoA SILEC Profile. Generate a m/z list from the neutral-loss scan and compare them against a m/z list of CoA species (supplementary table ); prepare a SRM experiment for each of these parent ions and their respective

product ions. The singly charged protonated molecule should be used for the parent CoA, whereas the associated product ion of 507 amu lower than the parent protonated molecule should be used as the SRM product ion. For example, acetyl-CoA

has a parent protonated molecule of m/z = 810.1 and a product ion at m/z = 303.1, whereas [¹³ C₃ ¹⁵ N]-acetyl-CoA has a parent protonated molecule of m/z = 814.1 and a product ion at m/z = 307.1.

(19)Determining labeling efficacy. Reinject and analyze the sample using the LC-SRM/MS method, monitoring both the labeled and unlabeled channels to determine the amount of unlabeled CoA remains for each acyl-CoA.

(20)Confirmation of acyl-CoA species. To confirm the identity of the labeled CoA species, spike extracted SILEC internal standards with a mixture of the CoA thioesters of interest (1 pmol each) and repeat the LC-SRM/MS analysis. The labeled and unlabeled compounds should co-elute. This is particularly important when multiple peaks arise or if there are multiple isobaric CoA species with similar parent and product ions.

(21)If labeling is complete ( > 99% labeling) and CoA profile is satisfactory, perform CoA extraction for all plates (Steps 6–9).

(22)Pool together acid-soluble SILEC extracts, label the batch and freeze at − 80 °C in 10-ml aliquots.

Generating customized sIlec standards

(23) (Continued from Step 21) Wash half of the plates twice with PBS.

(24) For each of the washed plates, replace with 10 ml of customization medium (see REAGENT SETUP) and incubate for 1 h.

(25)Test labeling using Steps 17–20.

(26) After incubation, harvest and extract CoA from all plates (Steps 6–9) and pool extracts together. This pooled extract should contain a SILEC CoA profile with increased levels of the species of interest.

producing purified [ ¹³ c₃ ¹⁵ n]-coasH

(27)(Continued from Step 5) After overnight incubation in ultra-labeling medium, perform Steps 6–9 for all plates and pool together acid-soluble lysates, which contain CoASH and short-chain acyl-CoAs.

(28)Adjust the pH of the lysate to ~13 with 40% (wt/vol) KOH. Add 500 µl of KOH to 5 ml of TCA extract and incubate at room temperature for 1 h.

(29)Adjust the pH of the lysate to 6 with formic acid.

(30) Purify the labeled CoA using SPE extraction (Steps 10–16).

(31)Dry down the hydrolyzed extract, which contains purified [¹³ C₃ ¹⁵ N]-CoASH. This product can now be used to chemically synthesize a particular CoA derivative.

(32)Thaw an adequate amount of previously generated acid-extracted SILEC CoA internal standard mixture. Generally, 1 ml of internal standard mixture is needed for every five samples. After thawing, vortex to ensure that the sample is mixed thoroughly and keep it on ice.

(33)To prepare standard curve samples, thaw and mix equal concentrations of unlabeled CoA standards of interest, creating a master mix, and perform twofold serial dilutions, from 1 µM down to ~10 nM, in extraction solution (10% (wt/vol) TCA).

Mix 50 µl of each dilution with 0.75 ml of extraction solution and 0.2 ml of thawed SILEC CoA internal standard extract (for a final volume of 1 ml) and vortex. This will generate a standard curve from approximately 100 fmol to 10 pmol of each CoA standard on the column.

(34) To prepare experimental samples, add 0.8 ml of ice-cold TCA along with 0.2 ml of the same SILEC internal standard mixture to harvested cell pellets, snap-frozen or lyophilized tissues, or purified extracted cells (platelets, lymphocytes, fibroblasts and so on.)

(35)For cell samples, sonicate for 30 s on ice in a pulsatile manner. For tissue samples, homogenize according to standard protocols. In either case, perform the same processing step for the standard curve samples as well.

(36)Spin processed acid-extracted lysate at 15,000g for 5 min at 4 °C to precipitate the protein pellet.

(37)Perform SPE extraction of CoA species as previously described (Steps 10–16).

(38) Resuspend the samples in 50 µl of 5% (wt/vol) SSA.

(39) For analysis, inject 10 µl from each sample or standard mix and perform LC-SRM/MS analysis using the method developed previously.

(40) From the raw data, develop a standard curve for each of the CoA species, with the x axis as the known amount of injected standard and the y axis as the ratio of unlabeled to labeled integrated peak areas.

(41)Use the regression line from this standard curve to calculate the amount of each CoA thioester in the samples

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