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Supporting Table 1

Table 1. A summary of the mass spectrometry analysis of AceCS1 and AceCS2

The tryptic digests of AceCS1 and AceCS2 and their corresponding acetylated forms were analyzed by MALDI-TOF and nanospray LC-MS/MS. The MALDI data peptides were identified by peptide mass fingerprinting using Protein Prospector while the LC-MS/MS data was analyzed using Mascot.

Supporting Text

Briefly, the gel pieces were cut into 1 mm cubes and washed with nanopure water. They were then destained using 100 mM (NH₄)HCO₃/50% methanol before being dehydrated step-wise in 25 mM (NH₄)HCO₃/50% acetonitrile and 100% acetonitrile. Gel pieces were then dried using a speed-vac before being rehydrated in 25 mM DTT in 25 mM (NH₄)HCO₃ for 20 min at 55°C. The DTT solution was then discarded and the gel pieces were alkylated in 55 mM iodoacetamide in 25 mM (NH₄)HCO₃ for 20 min in the dark at room temperature. The liquid was again discarded and the gel pieces washed with nanopure water. The dehydration and drying steps were repeated before the pieces were rehydrated in 20 ng/ml trypsin in 25 mM (NH₄)HCO₃ in 3% acetonitrile. The pieces were covered in 25 mM (NH₄)HCO₃ and incubated at 37°C overnight. One aliquot of the tryptic digestion was purified using a C18 ziptip and analyzed by MALDI-TOF and MALDI-TOF-TOF while another was analyzed by nanospray LC-MS/MS.

For the MALDI analysis, the sample was spotted using a matrix of 10 mg/ml a-cyano-4-hydroxycinnamic acid in 10 mM ammonium phosphate/50% acetonitrile and analyzed in positive mode.

For the LC-MS/MS analysis, the sample was acidified before being loaded onto a Zorbax 300SB-C18 5 mm x 0.3 mm cartridge for 15 min in 0.1% formic acid in water. The samples were then switched onto a 150 mm ´ 75 mm 300SB-C18, 3.5 mm Zorbax column that was in-line with a New Objective pulled fused silica ESI emitter. Gradient elution was performed at a flow rate of 0.28 ml/min using 0.1% formic acid in water (solvent A) and 0.1% formic acid/95% acetonitrile in water (solvent B). The gradient was held at 5% B for 15 min before ramping to 50% B over 85 min. The column was then ramped to 100% B over 15 min and returned to 5% B over 5 min. The column was reequilibrated in 5% B for 25 min. The MS was operated with by acquiring a survey scan over the m/z range 300-2200 with 4 spectra averaged per scan. From each survey scan, four precursors were selected automatically for MS/MS. Precursors were isolated with a 4-amu window. Dynamic exclusion was enabled for precursor selection with a repeat count of 2. For each MS/MS precursor, five spectra were averaged to generate the final spectrum.

The nanospray LC-MS/MS analyses for both protein sets did not identify the acetylated peptides but did show a lack of non-specific acetylation at other sites (see Table 1).

The following primers were used in the cloning of AceCS1, AceCS2, SIRT1, SIRT3 SIRT5 and SIRT6

AceCS1

Forward – 5'-TAAAGCTTCTGGGTGGTCAGGCAGCGGTGACT-3'

Reverse – 5'-ATGAGCTCATGGGGCTTCCCGAGGAGC -3'

AceCS2 full length

Forward – 5'-TGGATCCATGGCGGCCGCAGCCTCG-3'

Reverse – 5'-AAGTCGACCGTTGGTAGCAGCCCGCTGCTCT -3'

AceCS2 truncated

Forward – 5'-TGGATCCCCGGGCTGCGTTCCT-3'

Reverse – 5'- AAGTCGACCGTTGGTAGCAGCCCGCTGCTCT -3'

SIRT1

Forward – 5'-GCTTGGGATCCATGGCGGACGAG-3'

Reverse – 5'-CTCGAGTCGACATGATTTGTTTGATGGATAGTTCAT -3'

SIRT3

Forward – 5'-CCGGGATCCAAGGGGAAGCTTTCCCTGCAG-3'

Reverse – 5'-CTCGAGTCGACATTTGTCTGGTCCATCAAG-3'

SIRT5

Forward – 5'-ATAGGAATTCTGATGCGACCTCTCCAGATTGTCCCA-3'

Reverse – 5'-CCCAAGCTTAGAAACAGTTTCATTTTCATG-3'

SIRT6

Forward – 5'-CCGGGATCCATGTCGGTGAATTACG-3'

Reverse – 5'-CCCAAGCTTGCTGGGGACCGCCTT-3'

The plasmids were transformed into Escherichia coli BL21DE3 and grown in 2´ YT containing 100 mg/liter ampicillin at 37°C. Once growth reached an OD of 0.4-0.7 at A₆₀₀, isopropyl-D-thiogalactopyranoside (IPTG) was added and the protein expression of either SIRT1, SIRT3, SIRT5, SIRT6, AceCS1, or AceCS2 was induced for 8 h at 25°C. Cells were harvested and lysed by sonication in 50 mM Tris·HCl (pH 8), 250 mM NaCl, 5 mM imidazole, 1 mM 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and 5 mg/ml aprotinin. Cell debris was removed by centrifugation. The supernatant was agitated with nickel-nitrilotriacetic acid resin for 1 h at 4°C. The resin was then loaded onto a column and washed with 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, and 1 mM 2-mercaptoethanol. The protein was eluted with a linear gradient of 0-150 mM imidazole in 50 mM Tris·HCl (pH 8.0), 250 mM NaCl, and 1 mM 2-mercaptoethanol. Eluted protein was pooled, concentrated and dialyzed in 25 mM Tris (pH 7.5), 100 mM NaCl, 10% glycerol (wt/vol), and 5 mM DTT and stored at -20°C before use. AceCS1 and AceCS2 were further purified. The proteins were concentrated and dialyzed into 30% (NH₄)₂SO₄, 50 mM Tris·HCl (pH 8.0), 1 mM DTT, and 10% glycerol. AceCS1 and AceCS2 were then applied to a phenyl-Sepharose HP (Amersham Pharmacia) column (5 cm ´ 5 cm) equilibrated with 30% (NH₄)₂SO₄, 50 mM Tris-HCl (pH 8.0), 1 mM DTT, and 10% glycerol. The column was eluted with a decreasing gradient of 30-0% (NH₄)₂SO₄ in 50 mM Tris·HCl (pH 8.0), 1 mM DTT, and 10% glycerol. Active fractions were concentrated, and stored as described.