4800 maldi tof tof analyzer

Total cellular fatty acid methyl ester (FAME) profiles of strain IIIJ3-1 and its closest phylogenetic neighbors (type strains) were determined by the growing cells of respective strains on LB medium for 24h at 30 °C [101 (link)]. Cellular fatty acids were saponified, methyl-esterified, and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The FAMEs were analyzed using Gas Chromatograph (GC, Clarus 500, PerkinElmer) and compared with standard bacterial acid methyl ester mix (BAME, Sigma) for their identity. Isoprenoid quinines were extracted following the protocol mentioned by Dispirito et al. [102 ] separated using a SB-C18 Zorbax reverse-phase column fixed to a High Pressure Liquid Chromatograph (1100 Series, Agilent) with a solvent system of methanol: isopropanol (75:25 v/v) maintaining a flow rate of 1ml/min. Fractions corresponding distinct peaks at 2.9, 4.3 and 8.3 minutes were collected and concentrated in an evaporator. Molecular masses of the constituent quinones were analyzed by 4800 MALDI TOF/TOF analyzer (Applied BiosystemsInc., USA) using sinapinic acid as matrix. Constituent menaquinones were analysed by comparing the molecular masses in NCBI PubChem (https://pubchem.ncbi.nlm.nih.gov/).

The conversion of tandem switch for 5- and 6-membered O-S acyl shift was conducted in 2–3 mg peptides within 1 mL total volume including TFA, 0.05%–1.5% TfOH as a freshly pre-made solution of 10% TfOH/TFA by volume, and 5% thiocresol (50 µL) at room temperature for 2 h, 4 h, and more. The remaining thiocresol was removed by ether precipitation twice. The yield of all peptide-TC products was obtained by analytical RP-HPLC purification on a Shimadzu 10 series module with Diode Array UV detection, using a Vydac C18 column, with a linear gradient of 2% buffer B to 100% buffer B for 40 min and retained for 10 min in 100% buffer B after 40 min (Buffer A = 0.05% TFA in water; buffer B = 0.045% TFA in 60% acetonitrile in water). However, the area percentage of HPLC for other side products of small amounts such as “before 22 min” and “after 34 min” was employed (Supplementary Materials Table S1). The molecular weight of all products was confirmed by 4800 MALDI TOF/TOF Analyzer from Applied Biosystems.

Electrospray ionization (ESI) mass spectra were recorded using a bench-top ion trap mass spectrometer (FINNIGAN LCQ Deca XP MAX) equipped with standard ESI sources.
Mass of protein was recorded on a 4800 MALDI TOF/TOF Analyzer from Applied Biosystems which uses tandem time-of-flight technology for tandem mass spectrometry and is designed for sensitive, high throughput protein identification. The mass spectra were obtained in the MS reflector positive ion mode and using sinapic acid as the matrix.

Dried roots, seeds, and flowers from Panax ginseng, P. quinquefolius, or P. notoginseng (Yue Hwa Chinese Products Emporium Ltd., Singapore) were pulverized and 100 mg were extracted with 0.5 mL 50% ethanol to screen for CRPs with molecular masses of 2–6 kDa by mass spectrometry using an Applied Biosystems 4800 MALDI TOF/TOF Analyzer. To obtain sufficient ginsentides for characterization studies, ~2 kg of dried material were extracted with 10 L water. The extracts were filtered and subjected to flash chromatography using C18 powder (Grace Davison). The ginsentide-enriched fractions were subsequently eluted with 60% ethanol and concentrated using a rotary evaporator. The concentrated fractions were then purified by preparative RP-HPLC using a C18 Grace Vydac column (250 × 22 mm) at a flow rate of 8 mL/min on a Shimadzu system. A linear gradient of 1%/min of 10–80% buffer B was applied. Buffer A contained 0.05% (v/v) trifluoroacetic acid (TFA) in HPLC grade water, and buffer B contained 0.05% (v/v) TFA and 99.5% (v/v) acetonitrile (ACN). To obtain isolated ginsentides, the resulting fractions were further purified by a semi-preparative C18 Vydac column (250 × 10 mm), using the same gradient, at a flow rate of 3 mL/min.

All amino acids and resins were purchased from AAPPTec LLC (Louisville, KY, USA), Chem-Impex International Inc. (Wood Dale, IL, USA), and CEM Corporation (Matthews, NC, USA). All organic solvents were purchased from Millipore Sigma Corporation (St. Louis, MO, USA), Fischer Scientific (Pittsburgh, PA, USA), and Gyros Protein Technologies, Inc (Tucson, AZ, USA). Anticancer agents Doxorubicin (Dox) and Docetaxel (Doce) were purchased from LC laboratories (Woburn, MA, USA). Masses of intermediate and final products were confirmed by high-resolution matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometer from Bruker Inc. (GT 0264, Billerica, MA, USA) or Applied Biosystems (4800 MALDI TOF/TOF Analyzer, Foster City, CA, USA). Intermediate and final compounds were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) from Shimadzu (Prominence, Columbia, MD, USA) using a gradient system of acetonitrile and water with 0.1% trifluoroacetic acid using reverse phase C18 column (XBridge BEH130 Prep C18), from Waters Corporation (Milford, MA, USA). 5(6)-Carboxyfluorescein diisobutyrate (CFDI) was used to synthesize fluorescently-label peptide (USBiological Life Science, Swampscott, MA, USA).