This dataset contains raw and processed gas chromatography–mass spectrometry (GC-MS) output files generated from the analysis of a trimethylsilyl (TMS)-derivatized biological sample (designated "CASSI SAMPLE” 1,2 and 3) subjected to 0, 1.5, 3, and 5-hour hydrolysis, acquired on December 31, 2021, and quantitated April 14, 2025. SAMPLE 1 is carbohydrate preparation (see methods) precipitated with 5 % ethanol. SAMPLE 2 is a carbohydrate preparation precipitated with 70 % ethanol. SAMPLE 3 is the clarified lysate of HB101 Escherichia coli, from which SAMPLE 1 and 2 are from (see methods).  The samples were introduced via a split injection (20:1 ratio) using an Agilent 7890 GC system (serial no. CN13181037) equipped with a DB-5ht capillary column (30 m × 0.25 mm; J&W 122-5731) with helium as the carrier gas, coupled to a mass selective detector (MSD1). The analytical method ("LD E coli SUGAR for Cassi 52MINS_122621.M") was designed for carbohydrate profiling in a biological matrix consistent with microbial (E. coli) origin, with a 51.67-minute total run time and a multi-ramp oven program spanning 40 °C to 375 °C. This dataset is suitable for several research and educational applications: 1) Microbial lipid and carbohydrate profiling studies, particularly those focused on E. coli or similar gram-negative organisms; 2) Method benchmarking for TMS derivatization protocols and GC-MS sugar analysis pipelines; 3) Chemometrics and peak deconvolution training, given the complex chromatographic region from RT 30–50 min; or 4) Instrument performance assessment, as the cnorm.ini file provides calibration normalization data across four tune types (atune, air, BFB_Atune, lomass). This dataset contains no human subject data and presents no identifiable personal information. The analytical method files include proprietary Agilent software infrastructure (MSD ChemStation); users seeking to reprocess raw data may require compatible licensed software. No special biosafety, export control, or ethical review restrictions are apparent.

GC–MS Analyses of Derivatized Sugars

Gas chromatography-mass spectrometry (GC-MS) analysis was performed using an Agilent 7890A GC, coupled with a 5975C MS detector (MSD) capable of scanning up to 1000 m/z. The system utilized electron impact (EI) ionization at an approximate voltage of 1900 V, with data acquisition and analysis conducted using ChemStation software (Agilent Technologies). Prior to analysis, bacterial carbohydrate samples underwent trifluoroacetic acid (TFA) hydrolysis (2M) for varying durations of 1.5, 3, and 5 hours to release monosaccharides. Following hydrolysis, samples were derivatized using trimethylsilyl (TMS) reagents to enhance volatility for GC-MS analysis. Derivatization was carried out by adding 1- (Trimethylsilyl)-1 H-imidazole (TMSI) Silylation Reagent (Thermo Fisher Scientific), followed by incubation at 80 °C for 30–60 minutes. After cooling to room temperature, the samples were injected into the GCMS system at 270 °C using a 20:1 split mode for analysis. 

For gas chromatographic separation, a DB-5HT MS column (30 m × 0.25 mm ID × 0.25 µm film thickness with a maximum temperature limit of 400 °C) was used with helium (99.999 %) as the carrier gas at a flow rate of 0.6 mL/min. The oven temperature was programmed to increase from 40 °C to 375 °C, with the gradient optimized for effective separation. The mass spectrometer was operated with a scan range of 45–900 m/z. The ion source temperature was set to 230 °C, and the quadrupole temperature was maintained at 150 °C. Peaks were identified by comparing retention times to sugar standards and by matching the mass profiles of the sugar fragments against the NIST and Wiley spectral libraries, allowing for compound confirmation based on mass fragmentation patterns. Data analysis involved integrating peak areas and compiling them into a summary table, which was subsequently used to calculate the relative ratios of detected sugars.

E. coli Polysaccharide Purification

For biochemistry experiments, we used E. coli strain HB101 [supE44 hsdS20(rB-mB-) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1] and used the following protocol. HB101 was grown in 1 liter of Terrific Broth and shaken at 37 °C overnight. The next day the bacteria was spun down at 8,000 x g for 15 minutes. The pellet was washed four times with distilled water and resuspended in 50 ml ice cold PBS along with 100ul DTT (1M), 100ul Aprotinin (100x), 30ul Dnase1 (10mg/ml). The HB101 solution was then put through the LM20 high-pressure microfluidizer- 3 passes at 20000 PSI then 3 passes at 25000K PSI. Next, the HB101 homogenized cell lysate was put through a high-speed spin using a 45Ti rotor in an ultracentrifuge at 150,000-180,000 x g for 2 hours. After centrifugation both the supernatant and pellet were collected and stored at 4 °C. From the high-speed supernatant (now labeled ‘clarified lysate’), we performed an ethanol precipitation at -20 °C overnight. The final volume of ethanol was either 5 % or 70 %. The ethanol solution was spun in a microcentrifuge at 13.3 rpm for 10 minutes. After centrifugation of ethanol-treated solution, precipitates were collected and resuspended in 1x PBS at pH 7.4 at the same original volume of the high-speed spin supernatant used. To digest proteins, 800 units/ml of proteinase K (a broad-spectrum serine protease) was added to the resuspended ethanol precipitate for 1 hour at 37 °C. Protein digestion was confirmed by running samples on Any kD Mini-PROTEAN TGX Stain-Free protein gels. For DNA digestion, 10 units/ml of DNase 1, was added to the resuspended ethanol precipitate for 1 hour at 37 °C. For RNA digestion, 1 units/ml of RNase H was added to the resuspended ethanol precipitate for 1 hour at 37 °C.