Acid hydrolysis of silk fibroins and determination of the enrichment of isotopically labeled amino acids using precolumn derivatization and high-performance liquid chromatography-electrospray ionization-mass spectrometry.
Silk fibroins from moth larvae and spiders are composed of highly repetitive Ala- and Gly-rich blocks that determine their structure, properties, and function. To investigate the metabolic integration of isotopically labeled amino acids in the excreted silk, the enrichment of ingested tracers was determined after acid hydrolysis of the fibroins. Thus, spiders and moth larvae were fed with stable isotope tracers such as [1-13C]Ala or [1-13C]Gly and silked. After hydrolysis of the silk proteins, the corresponding amino acids were derivatized with Nalpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent) and separated by liquid chromatography. The isotopical enrichment of the amino acids was determined by online electrospray mass spectrometry and calculated by newly developed software. Depending on the feeding protocol, enrichments of up to 58% in Gly and 31% in Ala were found in the investigated silks. The highly enriched silk fibroins are suitable for further structural investigation such as solid-state nuclear magnetic resonance.
Hess
Sonja
S
Structural Mass Spectrometry Facility, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 8, Rm B2A27, Bethesda, MD 20892-0805, USA. Sonja_Hess@nih.gov
van Beek
Jacco
J
Pannell
Lewis K
LK
D019342:Acetic Acid / Q000737:chemistry; D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D012831:Bombyx / Q000737:chemistry; D002138:Calibration; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004032:Diet; D004136:Dinitrobenzenes / Q000737:chemistry; D005088:Exocrine Glands / Q000737:chemistry; D005349:Fibroins / Q000096:biosynthesis / Q000737:chemistry*; D006868:Hydrolysis; D019476:Insect Proteins / Q000096:biosynthesis / Q000737:chemistry*; D007814:Larva; D009590:Nitrogen Radioisotopes; D047011:Silk; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013112:Spiders / Q000378:metabolism
D000596:Amino Acids; D002250:Carbon Radioisotopes; D004136:Dinitrobenzenes; D019476:Insect Proteins; D009590:Nitrogen Radioisotopes; D047011:Silk; D005349:Fibroins; C047134:Marfey's reagent; D000409:Alanine; D019342:Acetic Acid