Amino acid analysis.
Amino acid analysis (AAA) is one of the best methods to quantify peptides and proteins. Two general approaches to quantitative AAA exist, namely, classical postcolumn derivatization following ion-exchange chromatography and precolumn derivatization followed by reversed-phase HPLC (RP-HPLC). Excellent instrumentation and several specific methodologies are available for both approaches, and both have advantages and disadvantages. This unit focuses on picomole-level AAA of peptides and proteins using the most popular precolumn-derivatization method, namely, phenylthiocarbamyl amino acid analysis (PTC-AAA). It is directed primarily toward those interested in establishing the technology with a modest budget. PTC derivatization and analysis conditions are described, and support and alternate protocols describe additional techniques necessary or useful for most any AAA method--e.g., sample preparation, hydrolysis, instrument calibration, data interpretation, and analysis of difficult or unusual residues such as cysteine, tryptophan, phosphoamino acids, and hydroxyproline.
Crabb
J W
JW
W. Alton Jones Cell Science Center, Lake Placid, New York, USA.
West
K A
KA
Dodson
W S
WS
Hulmes
J D
JD
D000096:Acetone; D000596:Amino Acids / Q000032:analysis*; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D003956:Dialysis; D004986:Ether; D006868:Hydrolysis; D006909:Hydroxyproline / Q000378:metabolism; D008567:Membranes, Artificial; D008698:Mesylates / Q000378:metabolism; D008872:Microwaves; D010670:Phenylthiourea / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis; D011145:Polyvinyls; D011506:Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D005465:Fluorocarbon Polymers
D000096:Acetone; D000596:Amino Acids; D004986:Ether; D006909:Hydroxyproline; D008567:Membranes, Artificial; D008698:Mesylates; D010670:Phenylthiourea; D010750:Phosphoproteins; D011145:Polyvinyls; D011506:Proteins; D013438:Sulfhydryl Compounds; C013976:dodecylmercaptan; C045880:methanesulfonic acid; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers