Mass spectrometry has suddenly expanded out of research and assay laboratories into biology, medicine and therapeutics. Electrospray ionization and matrix-assisted laser desorption/ionization yield increased mass-range and sensitivity, leading to novel applications and sparking new analyzer designs, software, and robotics. Though mass spectrometry is a century-old technique, only within the last two decades it has emerged as a tool for biomolecular analysis, first and particularly in the field of Proteomics, lately also in the field of Genomics.
Mass spectrometers equipped with ToF analyzers, first developed in the 1950s, separate ions according to their mass-to-charge ratio. Ions emitted from a source are accelerated so that those with like charge have the same kinetic energy. Those with a lower mass-to-charge ratio have a higher velocity than their higher mass-to-charge counterparts and thus reach the detector more quickly.
When paired with so-called hard ionization sources that break molecules into fragments during ionization, ToF analyzers have limited use in protein, peptide, and nucleic acid analysis. But, when coupled with two “soft” ionization methods that generate few fragment ions -- MALDI (matrix-assisted laser desorption ionization) and ESI (ElectroSpray Ionization) -- ToF systems can be used to analyze even large biomolecules. Developed in the mid-1980s by German scientists Franz Hillenkamp and Michael Karas together with Shimadzu researcher Koichi Tanaka, MALDI sources employ a chromophoric matrix in which the sample is dissolved; the matrix-sample solution is then dried on a target plate and exposed to UV laser light. The matrix absorbs energy from the laser and transfers this energy to the sample in the form of heat, which causes the sample to desorb (vaporize) and ionize.
Though MALDI-ToF mass spectrometers are common sights in most analytical molecular biology facilities, MALDI has still its limitations. Most notably, MALDI is a solid-state, pulsed technique that cannot easily be coupled online with liquid-based, continuous purification methods such as HPLC. For HPLC-based applications, ESI mass spectrometers are generally the tools of choice. First described by Northwestern University scientist Malcom Dole in the 1960s, ESI gained prominence as a method for biomolecular analysis in the late 1980s through the efforts of Yale University researcher John Fenn, who earned a shared 2002 Nobel Prize in Chemistry for his work in this field.
In ESI, a liquid sample is forced through a capillary tip in the presence of an electric field. As the liquid becomes charged, its molecules begin to repel each other, forming a fine mist of charged droplets. Solvent is evaporated using a neutral carrier gas, concentrating the charged analyte molecules into smaller droplets that then explode owing to repulsive forces between like charges. The process continues until analyte ions are completely stripped of solvent and only multiply-charged ions remain.
Fig. 1: Schematic illustration of
a) Electro Spray Ionization (ESI) technique and
b) Electro Spray Ionization (ESI) technique coupled to a TOF detector.
The resulting spectrum is comprised of peaks representing different charge states of the analyte.
Fig 2: Measured raw data of an oligonucleotide and calculated molecular weight past deconvolution.
Scientists analyzing (very) large molecules, such as proteins and nucleic acids, cite ESI’s ability to generate these multiply-charged ions as a major advantage, as it reduces the mass-to-charge (m/z) ratio of large ions, effectively extending the mass range of the spectrometer.
ESI mass spectrometers can be coupled to an HPLC system or interfaced with a nanoelectrospray apparatus. The latter alternative reduces the high flow and sample consumption rates typical of conventional HPLC but can be labor-intensive; nano-LC systems, though they employ lower flow rates, can be complicated to use even for the most experienced technician. To address these problems and in order to provide fit for routine high throughput applications e. g. reliably and significantly QCing oligonucleotides and DNA/RNA fragments, several integrated hard- and software solutions have been developed. metabion has decided to expand its mass spectrometry facilities into a defined HPLC-ESI-ToF solution, complementing its existing Maldi-ToF platform in order to improve analytical validity for long and complex oligonucleotides.
ESI-ToF technique was developed almost at the same time as MALDI-ToF. Compared to the MALDI-ToF method, ESI mass spectrometry displays some advantages in terms of resolution capacity of large/complex biomolecules as indicated above, hence optimizing the analytical/QC scope of metabion´s range of custom nucleic acid synthesis services.
Connection of the mass spectrometer to a HPLC system allows an improved analysis of complex samples as a result of the separation during HPLC on the one hand. On the other hand molecular weight determination of oligonucleotides in a wide range of 20 to more than 120 bases at high mass accuracy, resolution and sensitivity is possible, whereas MALDI-ToF – while being beyond doubt very accurate at lower MW ranges - is limited to significantly measuring oligonucleotides < 60 bases.
Expanding on above mentioned short description of the “ESI-ToF concept”, further more detailed information shall be adduced for consolidation and illustration of the aforesaid: Proceeding ionization of the molecules happens via deprotonation, whereas big molecules such as oligonucleotides can be multiply deprotonated, resulting in multiple negatively charged species (m/z = 2, 3, 4, 5, 6, …). The detected m/z values represent the raw data of the measurement and need to be processed applying a suitable deconvolution algorithm taking into account all measured peaks for calculation of the molecular weight of the measured sample.
Bringing amazing technologies forward from the academic level to industry by cost-effectively exploiting its power for routine applications in order to serve research and development “evolution” a difficult task. Metabion is trying to progressively, however deliberately and carefully accept this challenge . Optimizing hard- and software as well as biochemical requirements in order to meet our customers´ expectations in terms of added value to and additional benefit for increased operational efficiency is metabion´s driving force. Hence, our ambition of effectively introducing ESI-ToF as a routine QC-method where applicable follows our slogan: quality is primary, trust is principle.
References:
- J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong, C. M. Whitehouse, Science, 246, 1989, 64-71. Electrospray inonization for mass spectrometry of large biomolecules.
- M. E. Hail, B. Elliot, A. Anderson, Am. Biotechnol. Lab., 22, 2004, 12-14. High-troughput analysis of oligonucleotides using automated electrospray ionization mass spectrometry.
What we offer
ESI-ToF Service Measurements
If you have no possibility to measure long or complex oligonucleotides by ESI-ToF inhouse, we offer our profound experience in oligonucleotide mass spectrometry to help you elucidating your compound masses. You have the opportunity to choose ESI-ToF measurement without (w/o HPLC) or with (w HPLC) HPLC, which can be very useful for interpretation of complex oligonucleotide mixtures.
Do not hesitate to send us an aliquot of your sample either lyophilised (dry) or dissolved in water - metabion will supply you with the respective spectrum (for ESI-ToF measurement with HPLC you will also receive an analytical HPLC chromatogram of your sample).
Indication of the approximate amount and concentration, respectively of your sample and of the expected mass range would be helpful.
If you have a sample series or want us to measure your probes regularly please inquire for a quotation.
Prices
| ESI-ToF measurement | |
|---|---|
| Price € | |
| One sample w/o HPLC incl. spectral data | 35.00 |
| One sample with analytical HPLC incl. spectral data | 50.00 |
If you have a sample series or want us to measure your probes regularly please inquire for a quotation.