The Effect of Ammonium Salt and Matrix in the Detection of DNA by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Abstract

The effect of ammonium salt in the detection of oligonucleotides by matrix-assisted laser desorptionlionization time-of-flight mass spectrometry has been systematically investigated using several matrices with ammonium salt additives. The results show that the presence of ammonium salt in the matrix has a beneficial effect on protonation and deprotonation of oligonucleotides in addition to suppressing alkali-ion adducts. These observations indicate that a good matrix for DNA detection could be made from two components; i.e. one component for desorption, which has a relatively large absorption coeflicient at the incident laser wavelength, and another component for ionization, which has good protonation and deprotonation ability. Experimental results show that ammonium citrate and ammonium tartrate are good ionization components. Experimental results also indicate that large numbers of chemicals can be used as desorption components. The concept of a twocomponent matrix system opens many passibilities in the search for a good matrix to be used in the detection of biopolymers. The selection criteria for good matrices in this two-component system are discussed.The development of oligonucleotide detection using Matrixassisted laser desorption/ionization (MALDI) mass spectrometry has received great attention during the past several years due to its potential application in DNA sequencing. Tremendous effort has been put into the improvement of MALDI instrumentation and the search for more effective matrices. The former has led to several new techniques in M U D 1 instrumentation."* However, the results of extensive searches for new matrices, which are suitable for the detection of oligonucleotides, have fallen short of expectations. Good matrices have been limited to a few molecules, such as 3-hydroxypicolinic acid3' (3-HPA), picolinic acid4 (PA), and trihydroxyacetophenone? This slow progress in the search for effective matrices perhaps relates to the concept of what constitutes a good matrix. A good matrix generally should have two functions-desorption and ionization of an analyte upon laser irradiation. One often tries to search for a chemical compound which provides both of these functions, but results show that such chemical compounds are very few. On the other hand, little attention has been given to the possibility of constructing a good matrix by blending two components, chosen so that one component serves for desorption and the other for protonation/deprotonation. Because of this lack of attention, many areas have been left unexplored. Cume and Yates first pursued the possibility of a two-component matrix using an ammonium salt. They concluded that the addition of salts, such as ammonium acetate, to a UV-absorbing matrix can have a positive effect on the sensitivity, reproducibility, and mass range in the MALDI analysis of oligodeoxynucleotides. They also pointed out that ammonium salts may serve to stabilize the phosphodiester backbone in deoxyoligonucleotides such that self-protonation can occur. However, the ...

Oligonucleotide analysis with MALDI–ion-mobility–TOFMS

“…All matrices and additives, including 3-hydroxypicolinic acid (HPA) [31], 2,4,6-trihydroxyacetophenone (THAP) [32], anthranilic acid (AA) [33], 6-aza-2-thiothymine (ATT) [34], diammonium hydrogencitrate [35,36,37], and formic acid were purchased from Sigma (St Louis, MO, USA) or Aldrich Chemical (Milwaukee, WI, USA). External mass calibration was performed with a buckminsterfullerene mixture (C 60 with 10% C 70 ), also purchased form Aldrich.…”

Detection of Noncovalent tRNA·Aminoacyl-tRNA Synthetase Complexes by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

“…Therefore, we elaborated conditions for complex formation in volatile salt solutions known to be compatible with MALDI, e.g. ammonium salts which have been described as buffers and MALDI-matrix additives by several authors (25,29,41,42). Our final choice was 12.5 mM ammonium acetate in ultra-pure water, pH 7.…”

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry

“…Use of these stable homodeoxythymidine signal molecules also allowed for the use of ␣CHCA as the MALDI matrix, which cannot be used for the analysis of oligonucleotides containing mixed nucleotide sequences because of substantial backbone fragmentation of these oligonucleotides in this matrix (33). Use of ␣CHCA is advantageous because this matrix forms a more confluent, easily sampled, ''lawn'' of crystals on the MALDI probe tip than other MALDI matrices commonly used for DNA analysis.…”

“…Initial experiments (data not shown) showed that significant amounts of uncleaved probes are immobilized on the streptavidin-coated beads, eluted, and spotted on the MALDI probe tip along with the signal molecules. The presence of these longer probes in the MALDI-TOF MS analysis was a potential concern because of the fragmentation propensity of these oligonucleotides when ␣CHCA is used as the matrix (33). The resultant fragment ions could have m͞z values similar to those of the signal molecules produced in the secondary Invader reaction, leading to increased background levels and possibly yielding false-positive results.…”