Metabolite Profiling of Polar Extracts

The dried polar extracts were rehydrated with 600 µL 100 mmol L⁻¹ phosphate buffer in D₂O, pH 7.3, which included 1.0 mmol L⁻¹ TMSP (3-Trimethylsilyl 2,2,3,3-d₄ propionate, CAS 24493-21-8). Samples were reconstituted in sets of approximately 20 to minimize potential sample change/degradation from prolonged exposure to light and room temperature. The samples were analyzed using a Bruker Advance II 700 MHz spectrometer (Bruker Biospin, Inc., Billerica, MA) fitted with a cryogenically cooled probe (TCI 5 mm triple-resonance, z-gradient). Spectra were collected under automation with ICON-NMR with a one-dimensional (1D) ¹H and a two-dimensional (2D) ¹H J-resolved (JRES) pulse sequence. The 1D pulse sequence includes water suppression based on a three-pulse noesygppr1d which contains a spoiler gradient with 8 steady state scans, 40 transients, a 3 s relaxation delay, and a 60 ms mixing period. The data were acquired at 298 K with 65536 real data points across a spectral width of 20 ppm. The spectra were processed by multiplying the free induction decay by an exponential line broadening function of 0.3 Hz and the data were zero-filled to 65536 complex points prior to Fourier transformation (FT). The spectra were manually phased and the baseline was automatically corrected.
Two-dimensional edited heteronuclear single quantum correlation (HSQC) spectra with adiabatic ¹³C decoupling (HSQCEDETGPSISP2.2) were collected for selected samples to enhance metabolite identification. A relaxation delay equal to 1.5 s was used between acquisitions and a refocusing delay of 3.45 ms was implemented. In general, 2048 data points with 128 scans per increment were acquired with spectral widths of 11 ppm in F2 and 180 ppm in F1 (¹³C). The FIDs were weighted using a shifted sine bell function in both dimensions. Manual two-dimensional phasing was applied.
Shrimp metabolites were assigned based on 1D ¹H and 2D ¹H-¹³C NMR experiments. Assignments were based on comparison of chemical shifts and spin-spin couplings with reference spectra and tables such as those of the human metabolome database (HMDB) [31] (link), the Madison metabolomics consortium database [32] (link), the biological magnetic resonance data bank (BMRB) [33] (link), and an in-house compiled database, as well as the SBASE-1-1-2 and bbiorefcode_0_1_2 databases used with AMIX (version 3.9.11; Bruker Biospin, Inc., Billerica, MA) and Chenomx® NMR Suite profiling software (version 7.12).