Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome.
An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2 x 10(8) particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.
Lehmberg
E
E
Berlex Laboratories, Inc., Richmond, CA 94804-0099, USA. elisabeth_lehmberg@Berlex.com
Traina
J A
JA
Chakel
J A
JA
Chang
R J
RJ
Parkman
M
M
McCaman
M T
MT
Murakami
P K
PK
Lahidji
V
V
Nelson
J W
JW
Hancock
W S
WS
Nestaas
E
E
Pungor
E
E
D000260:Adenoviruses, Human / Q000737:chemistry*; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D020543:Proteome / Q000032:analysis*; D011786:Quality Control; D017421:Sequence Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019562:Viral Load; D014764:Viral Proteins / Q000032:analysis*; D015678:Viral Structural Proteins / Q000032:analysis
D000596:Amino Acids; D020543:Proteome; D014764:Viral Proteins; D015678:Viral Structural Proteins