Combination of a SAW-biosensor with MALDI mass spectrometric analysis.
A S-sens K5 surface acoustic wave biosensor was coupled with mass spectrometry (SAW-MS) for the analysis of a protein complex consisting of human blood clotting cascade factor alpha-thrombin and human antithrombin III, a specific blood plasma inhibitor of thrombin. Specific binding of antithrombin III to thrombin was recorded as a function of time with a S-sens K5 biosensor. Two out of five elements of the sensor chip were used as references. To the remaining three elements coated with RNA anti-thrombin aptamers, thrombin and antithrombin III were bound consecutively. The biosensor measures mass changes on the chip surface showing that 20% of about 400fmol/cm2 thrombin formed a complex with the 1.7-times larger antithrombin III. Mass spectrometry (MS) was applied to identify the bound proteins. Sensor chips with aptamer-captured (1) thrombin and (2) thrombin-antithrombin III complex (TAT-complex) were digested with proteases on the sensor element and subsequently identified by peptide mass fingerprint (PMF) with matrix assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry. A significant identification of thrombin was achieved by measuring the entire digest with MALDI-ToF MS directly from the sensor chip surface. For the significant identification of both proteins in the TAT-complex, the proteolytic peptides had to be separated by nano-capillary-HPLC prior to MALDI-ToF MS. SAW-MS is applicable to protein interaction analysis as in functional proteomics and to miniaturized diagnostics.
Treitz
G
G
Center of Advanced European Studies and Research (caesar), Ludwig-Erhard-Allee 2, D-53175 Bonn, Germany.
Gronewold
T M A
TM
Quandt
E
E
Zabe-Kühn
M
M
D000162:Acoustics / Q000295:instrumentation*; D000990:Antithrombin III / Q000032:analysis* / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D001774:Blood Chemical Analysis / Q000295:instrumentation* / Q000379:methods; D001798:Blood Proteins / Q000032:analysis*; D045424:Complex Mixtures / Q000032:analysis / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D010447:Peptide Hydrolases / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods
D001798:Blood Proteins; D045424:Complex Mixtures; C046193:antithrombin III-protease complex; D000990:Antithrombin III; D010447:Peptide Hydrolases