Precise mass measurement of a double-stranded 500 base-pair (309 kDa) polymerase chain reaction product by negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

摘要

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) has been used to determine the mass of a double-stranded 500 base-pair (bp) polymerase chain reaction (PCR) product with an average theoretical mass of the blunt-ended (i.e. unadenylated) species of 308 859.35 Da, The PCR product was generated from the linearized bacteriophage Lambda genome which is a double-stranded template, Utilization of ethanol precipitation in tandem with a rapid microdialysis step to purify and desalt the PCR product was crucial to obtain a precise mass measurement, The PCR product (0.8 pmol/ CLL) was electrosprayed from a solution containing 75% acetonitrile, 25 mM piperidine, and 25 mM imidazole and was infused at a rate of 200 nL/min, The average molecular mass and the corresponding precision were determined using the charge-states ranging from 172 to 235 net negative charges, The experimental mass and corresponding precision (reported as the 95% confidence interval of the mean) was 309 406 +/- 27 Da (87 ppm). The mass accuracy was compromised due to the fact that the PCR generates multiple products when using Tag polymerase due to the non-template directed 3'-adenylation, This results in a mixture of three PCR products with nearly identical mass (i.e. blunt-ended, mono-adenylated and diadenylated) with unknown relative abundances that were not resolved in the spectrum, Thus, the experimental mass will be a weighted average of the three species which, under our experimental conditions, reflects a nearly equal concentration of the mono- and di-adenylated species. This report demonstrates that precise mass measurements of PCR products up to 309 kDa (500 bp) can be routinely obtained by ESI-FTICR requiring low femtomole amounts, Copyright (C) 1999 John Wiley & Sons, Ltd. [References: 44]