Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) mass spectrometry (MS) is a common method used for quality control (QC) of oligonucleotides. MALDI-TOF instrumentation is suitable for use in high-throughput industrial QC settings and can resolve molecules in the size range of oligonucleotides. While small compared to nucleic acids from biological sources, synthetic oligonucleotides are nevertheless relatively large molecules, ranging in size from about 6000 daltons for a 20mer to 30,000 daltons for a 100mer. MALDI-TOF is useful for compounds up to ~15,000 daltons, or about a 50mer oligonucleotide. Samples above 15,000 daltons do not ionize and fly effectively and, additionally, are outside the optimal resolution range of these instruments. Electrospray Ionization-Time of Flight (ESI-TOF) MS can resolve longer oligonucleotides, up to 80 nt.
In the underlying MALDI technology the oligonucleotide test sample is mixed with a carrier or matrix (picolinic acid) and is deposited on a grid. The MALDI-TOF instrument uses pulses of laser light to vaporize the oligo/matrix in a process known as desorption. During this process, some molecules become ionized through protonation. The laser pulse can also fragment the molecule into a variety of charged and neutral particles (which can lead to spurious bands or artifacts in the resulting spectrum). In time of flight (TOF) mass spectrometry, ionized oligo molecules are accelerated by an electrostatic field in the mass analyzer to a common kinetic energy. With the same kinetic energy, lighter ions travel faster and heavier ions travel more slowly. The ionized particles enter at one end of the TOF tube, and the number of ions reaching a detector at the other end is recorded in a time-dependent manner. The lightest ions reach the detector first and the heaviest arrive last. The entire mass spectrum is recorded in a fraction of a second as ion flux versus time.
IDT pioneered the use of MS analysis of oligonucleotides, and throughout the years has provided MALDI-TOF MS traces as routine QC on all standard probes and primers as well as for more complex modified and HPLC and PAGE purified oligos. While many other oligonucleotide suppliers are just now starting to provide MALDI-TOF MS for their products, IDT has shifted our MS analyses to use of Electrospray ionization (ESI) instruments. As noted above, ESI technology maintains mass accuracy, resolution, and sensitivity over a broader nucleotide size range (Hail ME, Elliott B et al). The conversion from MALDI to ESI allows IDT to provide customers with greater mass accuracy for any oligo we synthesize up to 200 bases.
Reference: Hail ME, Elliott B, and Anderson A. (2004) High-throughput analysis of oligonucleotides using automated electrospray ionization mass spectrometry. Am Biotech Lab, 22: 12-14.