Kinetic binding analysis of aptamers targeting HIV-1 proteins by a combination of a microbalance array and mass spectrometry (MAMS).
An enhanced chip-based detection platform was developed by integrating a surface acoustic wave biosensor of the Love-wave type with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). The system was applied to characterize the interaction of aptamers with their cognate HIV-1 proteins. The aptamers, which target two proteins of HIV-1, were identified using an automated in vitro selection platform. For aptamers S66A-C6 and S68B-C5, which target the V3 loop of the HIV-1 envelope protein gp120, KD values of 406 and 791 nM, respectively, were measured. Aptamer S69A-C15 was shown to bind HIV-1 reverse transcriptase (HIV-1 RT) with a KD value of 637 nM when immobilized on the biosensor surface. HIV-1 RT was identified with high significance using MALDI-ToF MS even in crude protein mixtures. The V3-loop of gp120 could be directly identified when using chip-bound purified protein samples. From crude protein mixtures, it could be identified indirectly with high significance via its fusion-partner glutathione-S-transferase (GST). Our data show that the combination of the selectivity of aptamers with a sensitive detection by MS enables the reliable and quantitative analysis of kinetic binding events of protein solutions in real time.
Gronewold
Thomas M A
TM
Research Center CAESAR, Ludwig-Erhard-Allee 2, D-53175 Bonn, Germany.
Baumgartner
Antje
A
Hierer
Jessica
J
Sierra
Saleta
S
Blind
Michael
M
Schäfer
Frank
F
Blümer
Julia
J
Tillmann
Tina
T
Kiwitz
Anne
A
Kaiser
Rolf
R
Zabe-Kühn
Martin
M
Quandt
Eckhard
E
Famulok
Michael
M
D000595:Amino Acid Sequence; D052158:Aptamers, Peptide / Q000737:chemistry*; D019921:Biotinylation; D015699:HIV Envelope Protein gp120 / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014764:Viral Proteins / Q000737:chemistry*
D052158:Aptamers, Peptide; D015699:HIV Envelope Protein gp120; D010455:Peptides; D011506:Proteins; D014764:Viral Proteins; D054303:HIV Reverse Transcriptase