Peptide Nucleic Acid Characterization by MALDI-TOF Mass Spectrometry

Abstract

Peptide nucleic acids (PNAs) are a new class of DNA mimics in which the regular nucleobases of adenine, thymine, cytosine, and guanine are connected via a peptide-like backbone. PNA molecules retain the same Watson-Crick base pairing as regular oligonucleotides, with the added benefits of greater specificity and resistance to enzymatic digestion. While the use of PNAs has grown rapidly because of their potential applications in biotechnology, little work has been done on developing analytical procedures for characterizing them. We have found matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to be an effective tool for PNA analysis. PNA molecules survive the MALDI process intact and are easily ionized with almost no multiply-charged species. These features allow mixtures to be easily characterized. Traditional protein matrices (e.g., sinapinic acid,2,5-dihydroxybenzoic acid, alpha-cyano-4-hydroxycinnamic acid) were found to be superior to DNA matrices (e.g., trihydroxy-acetophenone, 3-hydroxypicolinic acid, picolinic acid). In addition, the new DNA matrix 6-aza-2-thiothymine worked well. The ability of MALDI-TOF-MS to ascertain PNA purity and sequence information at low picomole levels will be important as greater quality control of PNA synthesis is needed (e.g., when PNAs are used as antisense or antigene drugs).

Comprehensive nomenclature for the fragment ions produced from collisional activation of peptide nucleic acids

“…[6][7][8][9] Mass spectrometry (MS) has recently been employed for the characterization of PNA's. [10][11][12] Butler et al used matrix-assisted laser desorption/ionization (MALDI) coupled with time-offlight (TOF) MS to obtain PNA sequence information from intact masses of the synthesis byproducts that form from incomplete coupling of each monomer. 10 Griffith et al also demonstrated the use of electrospray ionization (ESI)-MS to determine the stoichiometry of PNA with DNA.…”

“…[10][11][12] Butler et al used matrix-assisted laser desorption/ionization (MALDI) coupled with time-offlight (TOF) MS to obtain PNA sequence information from intact masses of the synthesis byproducts that form from incomplete coupling of each monomer. 10 Griffith et al also demonstrated the use of electrospray ionization (ESI)-MS to determine the stoichiometry of PNA with DNA. 11 Takao et al reported the fragmentation pathways of PNA's in a highenergy collision cell (8 keV) using fast atom bombardment (FAB) as the ion source (vida infra).…”

Genotyping single nucleotide polymorphisms by mass spectrometry

“…These characteristics lead to greater specificity and selectivity than corresponding oligonucleotides. PNAs are very compatible with mass GENOTYPING SINGLE NUCLEOTIDE POLYMORPHISMS & spectrometric analysis because the backbone is charge neutral, and is thus insensitive to adduct formation and less prone to fragmentation (Butler et al, 1996). PNAs behave more like peptides, and are best analyzed with peptide matrices.…”

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry as a Rapid Quality Control Method in Oligonucleotide Synthesis

“…PNA has been demonstrated to ionize under MALDI conditions, but it appears that the traditional peak on CE being the fully phosphorylated and more highly charged species under denaturing conditions). protein matrices are superior to most of those used in DNA analysis (17). This suggests that the ionization efficiency, in nega-FIG.…”