Quantum dots improve peptide detection in MALDI MS in a size dependent manner
© Bailes et al; licensee BioMed Central Ltd. 2009
Received: 21 December 2005
Accepted: 31 December 2009
Published: 31 December 2009
Laser Desorption Ionization Mass Spectrometry employs matrix which is co-crystallised with the analyte to achieve "soft ionization" that is the formation of ions without fragmentation. A variety of matrix-free and matrix-assisted LDI techniques and matrices have been reported to date. LDI has been achieved using ultra fine metal powders (UFMPs), desorption ionisation on silicon (DIOS), sol-gel assisted laser desorption/ionization (SGALDI), as well as with common MALDI matrices such as 2,5-dihydroxy benzoic acid (DHB), 3,5-dimethoxy-4-hydroxycinnamic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA) to name a few. A variety of matrix additives have been shown to improve matrix assisted desorption, including silicon nanowires (SiNW), carbon nanotubes (CNT), metal nanoparticles and nanodots. To our knowledge no evidence exists for the application of highly fluorescent CdSe/ZnS quantum dots to enhance MALDI desorption of biological samples. Here we report that although CdSe/ZnS quantum dots on their own can not substitute matrix in MALDI-MS, their presence has a moderately positive effect on MALDI desorption, improves the signal-to-noise ratio, peak quality and increases the number of detected peptides and the overall sequence coverage.
The term 'MALDI' (matrix assisted laser desorption ionization) was first introduced by Karas et al. who documented the advantage of using a highly absorbing matrix that reduces the threshold irradiance required to generate ions in Laser Desorption Ionization Mass Spectrometry (LDI-MS). The presence of a matrix results in a larger degree of "soft ionization", that is the formation of ions without fragmentation. This soft laser desorption increases the ratio of molecular-to-fragment ions which is of great benefit in the detection of sample-specific ions. Matrix selection depends on the particular sample being analysed and can often be a case of trial and error to determine the one best suited, however it is typically a low molecular weight compound that is able to undergo phase transition upon excitation with laser. Because the matrix is co-crystallised with the analyte sample, this phase transition extends to the sample itself. Tanaka's "monumental blunder" when he unwittingly suspended his ultra fine metal powders (UFMPs) matrix in glycerol instead of acetone, and subsequently deviated from his standard protocol a further three times, only to stumble across a significant discovery  was an essential step and a breakthrough in the development of macromolecule ionization by laser irradiation. Typical chemical matrices used today are derivatives of UV absorbing organic acids such as benzoic, cinnamic or picolinic acids [3–8].
Sample excitation is usually achieved with short pulses of UV lasers in the wavelength range of 248-355 nm. Whilst 337 nm is the most commonly used wavelength for excitation with UV lasers, excitation with neodymium-doped yttrium aluminium garnet solid state Nd:YAG laser (frequency tripled to 355 nm) has been also reported for most of the common matrices. Spectrum quality generally increases with absorption and the best performance is often achieved when the excitation wavelength near matches that of the matrix absorption maxima. Dreisewerd provides a fine, extensive review on the topic for further reading .
Despite the application of MALDI to the analysis of a wide ranging catalogue of analytes and its relatively high tolerance of biological mixtures [10, 11], the technique still suffers from a number of inherent drawbacks. The low molecular weight nature of the matrix itself means low molecular weight compounds (below ~500 m/z) are difficult to analyze because their detection is masked by the generation and detection of matrix ions. The specificity requirements of the matrix method also mean that a laborious process of trial and error may be required to ascertain the best matrix for a particular sample, as well as the optimization of analyte:matrix ratios, and co-crystallisation to avoid the formation of "hot-spots" during sample deposition if electrospray equipment is not available. MALDI is also intolerant to salt. The quest to overcome these problems has led to the development of a new line of LDI mass spectrometry techniques that do not require matrices, techniques termed matrix-free LDI-MS.
Direct LDI-MS was initially examined on a range of surfaces [12–17], but results showed that the success of this method is highly dependable on the properties of the analyte, with a high level of molecular degradation resulting from the increased laser power required. Desorption ionisation on silicon (DIOS) harnesses two useful properties of porous silicon, its ability to absorb in the UV and its physical structure, capable of trapping analytes of interest on its surface. DIOS was first reported by Wie et al . who documents the generation of micrometer thick porous silicon layers from either n- or p-type flat crystalline silicon through an electrochemical etching process, in the presence of ethanol which helps to reduce background ion intensity.
In 2002 Lin & Chen  reported the development of a new technique dubbed sol-gel assisted laser desorption/ionization (SGALDI) mass spectrometry. The technique was shown to be compatible with small proteins, peptides, amino acids and small organics with detection limits stretching as far as 8.1 femtomoles. Chen & Chen  adopted a similar SGALDI principle to overcome sample deposition problems when using a 3,4-diaminobenzoic acid (DABA) and 3,5-DABA as matrices. Kinumi et al investigated eleven kinds of metal particle (Al, Mn, Mo, Si, Sn, SnO2, TiO2, W, WO3, Zn and ZnO) in an attempt to identify promising alternatives to organic matrices. The team analysed two analytes, PEG 200 and methyl stearate. Results were encouraging, with only one of the candidates, SnO2, unable to ionize both PEG 200 and methyl stearate. The most impressive results were obtained with TiO2 powder as the matrix suspended with liquid paraffin, with which both analytes exhibited their best signal:noise ratio.
In 2003, Xu et al . presented an interesting approach to MALDI analysis of biomolecules by using carbon nanotubes (CNTs) as a matrix. CNTs were discovered over a decade earlier  and have since been the subject of a wide range of experimental research. CNTs synthesized by Xu et al . displayed rod morphology with an overall diameter of ~20 nm each consisting of several cylindrical graphite sheets. CNTs not only absorb and transfer UV radiation to the analyte being studied, but also double as a good support for sample thus simplifying preparation procedures. A reduction in analyte fragmentation was also observed when CNTs are used as a matrix due to a lower fluence threshold. These factors, combined with the absence of any background ions from a CNT matrix, mean the method is highly useful for analysis of low molecular weight compounds, demonstrated by the successful analysis of organic compounds, β-cyclodextrin, and small peptides.
Given the surge of nanotechnology within the biological field, CNTs are unsurprisingly not the only nanostructures to be applied to MALDI analysis, silicon nanowires have also been used as a substrate for MALDI . In this report the strong fluid wicking properties of SiNWs that result from their high surface area were exploited, and the chromatographic separation was combined with subsequent LDI-MS analysis of metabolites in biological samples. As with other nanostructures, a lower laser power was required in order to generate ion detection. The most reproducible of all LDI-MS approaches involving nanoparticles is that of silicon nanocavities due to the non-random nature of their synthesis .
Quantum dots (QDs) are highly fluorescent inorganic semiconductor nanocrystals that possess a number of unique and exciting features. The peak emission of a QD is dependent on its physical size, meaning they can be tuned to emit at any given wavelength , whilst it is possible to excite all QDs simultaneously with only a single short excitation wavelength. The unique and superior photophysical properties of QDs have seen them incorporated into a wide spectrum of biological applications and non-biological technologies [27–33]. Given the considerable hype surrounding the potential for QDs to revolutionize numerous areas of science, it is perhaps little surprise that they also appear to offer assistance to one of biological science's mainstream analytical techniques, MALDI TOF-MS. Matrix compounds by definition must exhibit high absorption at the excitation source wavelength, a requirement that is certainly met by QDs, all of which absorb at any wavelength below that of their emission. The application of nanodots to LDI-MS has recently been reported by way of using self-assembled germanium nanodots (GeNDs) grown on a silicon wafer and used as a matrix free method of LDI-MS (GeND-MS) for detection of peptides, proteins, synthetic oligomers, and polymer additives . Useful mass spectra were obtained even for those masses under 800 m/z. Previous to this, platinum nanodots were incorporated into a novel silicon sample plate for MALDI-TOF-MS analysis of DNA , improving results and reproducibility. To our knowledge no evidence exists for the application of highly fluorescent CdSe/ZnS QDs to enhance MALDI desorption of biological samples.
Properties of CdSe/ZnS Core/Shell "EviDots" quantum dots from http://www.evidenttech.com unless specified otherwise
QD emission, nma
QD emission, nmb
Emission FWHM, nma,c
Estimated crystal diameter, nma
Estimated molecular weightd, g/mol
Approx. quantum yielda, %
where J0 is the zero-order Bessel function.
Quantum dot mixtures used to assist MALDI-TOF analysis
CdSe/ZnS Core/Shell "EviDots" quantum dots used (mixed 50:50% v/v)b
QD emissionof the mixtures, nmc
ED-C11-TOL-0520 (517 nm) + ED-C11-TOL-0540 (544 nm)
519 nm (Ex = 337 nm)d
520/539 nm (double peak, Ex = 380 nm)
ED-C11-TOL-0560 (566 nm) + ED-C11-TOL-0580 (579 nm)
575 nm (Ex = 337 nm)d
575 nm (Ex = 380 nm)
ED-C11-TOL-0600 (598 nm) + ED-C11-TOL-0620 (615 nm)
614 nm (Ex = 337 nm)d
614 nm (Ex = 380 nm)
1. Characterisation of quantum dot preparations
2. CdSe/ZnS quantum dots on their own can not substitute matrix in MALDI
3. The presence of CdSe/ZnS quantum dots in alpha-cyano-4-hydroxycinnamic acid matrix facilitates MALDI desorption, improves the signal-to-noise ratio and peak quality
4. The presence of CdSe/ZnS quantum dots in alpha-cyano-4-hydroxycinnamic acid matrix enhances peptide desorption and increase the number of detected peptides and the overall sequence coverage
Following the calibration (all spectra were calibrated using internal standards) and the de-isotoping, the peaks were automatically extracted using "FlexAnalysis" software and the mass-to-charge ratios (m/z) were submitted to the MASCOT search. Significantly more BSA masses were identified in all QD-containing samples. Results are summarised in Figure 4 (Panel C), which shows the maximum numbers of BSA masses identified by MASCOT from all spectra for each matrix/QD samples (the sequence coverage similarly increased, data not shown). Similarly to the S/N, QF and Res analyses described earlier, the values here are means from different experiments and the STDEV values shown indicate variability between the experiments and not between the values from different spectra for the same matrix/QD sample from the same experiment (the latter are highly variable since different laser power settings were used). The larger number of the identified masses resulted in noticeably higher MASCOT scores obtained for the samples containing QDs, see Figure 4 (Panel D). The use of QD2 matrix mixture yielded the highest MASCOT scores. In the absence of QDs, variability in the scores is significantly higher than in all other cases investigated. The "Expect" values reported by MASCOT (the probability that the match was a random event of no significance) were also different. The overall best (minimum) "Expect" value over all experiments, samples and spectra for the different QD samples was recorded for QD2 (p = 0.00014, i.e. over ×300 fold lower than the default Mascot value of 5%).
A number of additives are documented in the literature, the presence of which assists the matrix in achieving successful desorption/ionization of the analyte. Those mentioned so far include glycerol , used to ensure a uniform analyte matrix mixture is achieved and also aids release of the sample from its crystalline state, and NIPPOLAN-DC-205  used for immobilizing carbon nanotubes to the target plate. Glycerol has also performed well when used for sample desorption with infra-red solid state lasers (Er-YAG and Er-YSGG) . Paraffin was utilized in sample preparation by Kinumi et al for the analysis of small molecules. The inclusion of paraffin was shown to greatly reduce low molecular mass noise from matrix ions, which in turn permitted the improved study of small molecules with inorganic metal particle matrix. Cu(II) ions have also been successfully used as electron scavengers in MALDI to prevent the reduction of analytes that can impair analysis . Other additives include those such as ammonium citrate to improve spectral quality for protein digest analysis with DIOS . Platinum nanoparticles have been reported to improve MALDI results and  and self-assembled germanium nanodots (GeNDs) were shown to desorb peptides, proteins, synthetic oligomers, and polymer additives without any matrix added .
In our hands the colloidal dispersions of CdSe/ZnS Core/Shell "EviDots" QDs did not work without the matrix, but improved the performance of MALDI-TOF-MS when co-applied together with the alpha-cyano-4-hydroxycinnamic acid matrix. We can not be certain as to the exact mechanism of this effect and a number of explanations remain possible. On drying, the dots may have formed a sol-gel and increased the surface area at which the desorption occurred, thus increasing the efficiency of MALDI. Similar effects have been reported previously, e.g. SGALDI mass spectrometry using polymeric sol-gels with 2,5-Dihydroxybenzoic acid (DHB), 3,4-diaminobenzoic acid (DABA) and 3,5-DABA as matrices [19, 20, 47], or with titanium based sol-gels [48, 49]. Alternatively, the addition of QDs could have helped matrix crystallisation, by serving as nucleation centres for the growing matrix crystals. Another plausible explanation, from our point of view, is that the addition of QDs resulted in an adjustable red-shift of the 337 nm wavelength of the nitrogen laser and in the case of the best performing QD2 mix (with the Emission maxima at 561 nm and 579 nm (575 nm, when mixed at 50:50% v/v) could have improved the photon absorption by the alpha-cyano-4-hydroxycinnamic acid matrix. It has been reported that alpha-cyano-4-hydroxycinnamic acid matrix is suitable for use with both nitrogen laser (337 nm)  and the neodymium-doped yttrium aluminium garnet solid state Nd:YAG laser (355 nm) .
The maximum absorption for alpha-cyano-4-hydroxycinnamic acid in methanol is 340 nm (50 nm full width at half maximum) and the absorption peak does not extend into the area where main emission peaks of the "EviDots" are. At the same time no noticeable Emission was detected for any of the "EviDots" preparations below ~450 nm. However, when crystallised both the alpha-cyano-4-hydroxycinnamic acid absorption properties as well as QDs fluorescent properties could have changed. In our experiments we used nitrogen laser (337 nm) and the addition of QDs must have red-shifted the laser energy and the broader illumination spectrum must have better matched the absorption spectrum of the crystallised matrix. Clear distinction between the three QD preparations with the QD2 mixture (575 nm emission wavelengths) having the strongest effect supports the size and/or wavelength specific QDs effect on MALDI desorption. It is not impossible that all the above factors acted in concert and we do not exclude the possibility that another additional mechanism exist by which ED-C11-TOL-0560 and ED-C11-TOL-0580 "EviDots" specifically enhance MADLI desorption.
Here we report that CdSe/ZnS QDs have a moderately positive effect on MALDI desorption of crude tryptic digests by improving the signal-to-noise ratio, peak quality and increasing the number of detected peptides and the overall sequence coverage. CdSe/ZnS QDs on their own can not substitute matrix in MALDI-MS as no spectra were obtained in the absence of alpha-cyano-4-hydroxycinnamic acid matrix. We conclude therefore that the use of fluorescent quantum dots in addition to standard MALDI matrix may further improve the technique of MALDI-TOF-MS and extend the range of usable matrices. However, further work might be required to optimise the solvents, QD composition, QD-to-matrix ratios and QDs emission wavelengths
We thank HFL Ltd. for their financial support in the purchase of Evident quantum dot products.
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