A highly efficient and automated method of purifying and desalting PCR products for analysis by electrospray ionization mass spectrometry.
In this work we present a rapid and fully automated method to purify and desalt PCR products prior to analysis by electrospray ionization mass spectrometry. The protocol employs a commercial pipette tip packed with an anion-exchange resin and comprises four primary steps: tip pretreatment, sample loading, rinsing, and sample elution. This tip-based purification/desalting protocol has two distinct advantages over previously published methods. First, the protocol can be performed either manually (1-12 samples at a time), using a standard p10 manual pipette, or in a fully automated microtiter plate format (96 samples at a time) employing standard laboratory robotics. Additionally, the entire protocol from crude PCR product to an "electrosprayable" analyte solution requires only 10 microl of crude product and takes less than 20 min. Using capillary gel electrophoresis, we demonstrate an overall recovery efficiency of approximately 80% and demonstrate the exquisite desalting efficiency with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Using an internal mass standard we demonstrate sub-ppm mass measurement error which provides an unambiguous base composition for a 120-mer PCR product.
Jiang
Yun
Y
Ibis Therapeutics, A Division of Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008, USA.
Hofstadler
Steven A
SA
D001412:Bacillus subtilis / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D016680:Genome, Bacterial; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D004269:DNA, Bacterial