**4.3.3 Choice of matrix**

348 Gel Electrophoresis – Advanced Techniques

analyzed with MALDI-TOF MS with the same settings and with the laser induced collection of spectra in an automatic mode. The spectra were then used for NCBI database search with MS-fit using the same settings for all samples. All 17 proteins were identified with peptide mass fingerprinting in both ziptip cleaned and untreated samples. As shown in Table 2, ziptip cleaning significantly improved the peak intensities, signal to noise ratio and the mass accuracy. This illustrates that removal of salts and other contaminants that will compete with the peptides in the spectra increases the intensities of the peptide peaks and thereby increases the accuracy of the mass determinations and, as a consequence, also increases the reliability of the identifications. On the other hand, the number of peptides and sequence coverage found in ziptip cleaned samples were about the same as in the untreated samples (Table 2). In general, there was a clear tendency that in ziptip cleaned samples more peptides were detected in the lower mass region (<1000 m/z) while fewer peptides were detected in the higher mass region (>2000 m/z). This suggests that the removal of salt ions and low molecular chemicals with subsequent improved signal to noise increases the possibility to detect low molecular mass peptides but that this beneficial effect is counteracted by adsorption of larger peptides to the solid phase of the ziptip. To test this, 10 protein samples were sequential eluted from the ziptip with increasing ACN concentration, up to 90 %. Indeed, this procedure increased the number of peptides found and the sequence coverage increased from 52 +/- 16 % in the untreated samples to 58 +/- 16 in the ziptip cleaned samples (p<0.05). The effect varied among the different proteins but was in some samples quite profound, almost 2 times higher sequence coverage. It can be concluded from these experiments that purification of peptide samples with ziptip improves the results with MALDI-TOF MS. However, when it comes to the identification of proteins with peptide mass fingerprinting the beneficial effect of the cleaning procedure is quite limited as the number of peptides found, using the standard protocol, is not increased. Therefore, considering the work-load needed for the ziptip procedure, it is doubtful if it is practical to routinely clean samples with ziptip before MALDI-TOF analyses. However, for selected, low abundant, samples it can most likely make a significant difference for the identification. In these cases, sequential elution of the peptides from the ziptip with increasing acetonitrile

concentrations is recommended.

by Wilcoxons signed rank sum test.

**Without Ziptip** 

**Statistical** 

**Average error (ppm)** 

fingerprinting data obtained with MALDI-TOF MS.

**Sequence coverage (%)**

Table 2. Influence of sample cleanup of in-gel digested proteins on peptide mass

**Number of peptides** 

**(n=17)** 11.2 +/- 3.6 24.9 +/- 12.0 8 +/- 4 100 +/- 130 3500 +/- 2000 **Ziptip (n=17)** 7.9 +/- 4.4 24.1 +/- 11.4 8 +/- 3 200 +/- 480 6300 +/- 4000

**significance** 0.01 No No <0.001 <0.001

Proteins were separated by 2-DE and in-gel digested by trypsin. The same peptide samples were then purified by ZipTip, mixed with the matrix and spotted on the MALDI plate or directly mixed with the matrix and spotted on the plate. Statistical interpretations were done

**Signal/noise** 

**ratio Peak Intensity**

To enhance the quality of the MALDI-TOF mass spectra and the number of desorbed peptides there are several matrices that could be considered to use. Different matrix compounds, both acidic and basic, have proved to work in sample preparation for MALDI mass analyzers. The far most commonly used matrix for peptide mass fingerprinting is CHCA, which is recommended for peptides with mass ions below 2500 Da (Beavis et al., 1992). Alternative matrices also used are sinapinic acid, mostly for masses higher than 25 kDa (Lewis et al., 2000) and DHB, originally suggested for glyco- or phosphopepides that are difficult to ionize (Strupat et al., 1991), but later also proven useful for silver stained proteins (Ghafouri et al., 2007). As illustrated in figure 11, CHCA and DHB have very different crystal structures on the target plate. Whereas CHCA usually has a homogeneously distributed spot appearance, making it ideal for automatic laser induced peptide desorption, the DHB crystals are needle shaped and often aggregated into fan-like structures directed from the outside towards the centre of the sample spot. Interestingly, we have found that peptides appear to be enriched in the base of the DHB structures (figure 11), significantly increasing the signal to noise ratio in spectra obtained from these areas. This is illustrated by the identification of transthyretin, one of the proteins that interact with silica nanoparticles and carbon nanotubes (figure 5). With DHB, the improved signal to noise in the spectrum displayed twice the number of peptides than with CHCA (figure 12). This dramatically increased the sequence coverage from 39 % obtained with CHCA to 72 % obtained with DHB. Thus, the use of DHB for low-abundant silver stained proteins from 2- DE is recommended.

Fig. 11. Crystals of α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB).

A; CHCA and B; DHB as matrix on a MALDI plate. The marked area indicates the position of the laser where the best signal to noise was obtained with DHB.
