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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
Author(s) -
Muddiman David C.,
Allison P.,
Hannis James C.
Publication year - 1999
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(19990630)13:12<1201::aid-rcm643>3.0.co;2-x
Subject(s) - chemistry , fourier transform ion cyclotron resonance , electrospray ionization , mass spectrometry , analytical chemistry (journal) , chromatography , selected ion monitoring , sample preparation in mass spectrometry , selected reaction monitoring , tandem mass spectrometry , gas chromatography–mass spectrometry
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/µL) 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 Taq 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 di‐adenylated) 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 © 1999 John Wiley & Sons, Ltd.