4. Chromatographic separation of digested proteins

Upon tryptic digestion of proteins, resulting peptides are being separated on a chromatographic column prior to mass spectrometric detection and subsequent bioinformatics analysis. Separation of peptides is being performed either using one-dimensional approach or the multidimensional separation by combining two or more separation technologies prior to MS detection and analysis.

For the one-dimensional chromatographic separation approach, peptides are being injected onto the reversed phase nano HPLC column where they are separated according to their hydrophobic interaction with the stationary phase [60]. Figure 2 shows an exemplary base peak chromatogram (BPC) for one-dimensional analysis separation of salivary peptides. Usually, long separation gradients are selected for one-dimensional separation in order to provide the best possible conditions for peptide separations and large number of identifications.

Figure 3. Base Peak Chromatogram (BPC) of GluC-tryptically generated peptides, which were separated on a reversed phase nano HPLC column.

The separation shown in Figure 3 was performed using a 180-min gradient and a total analysis runtime of 210 min. Thus, the total amount of available time must be considered when performing this kind of analysis.

Enzymatic digest of salivary proteins does not differ from enzymatic digests used for other biological samples. Based on previous experience and results, trypsin is the most widely used protease for protein digest resulting with peptides suited for subsequent separation using cation exchange columns or anion exchange columns, reversed phase separation, and mass spectrometric detection. A comparison of results obtained using different digestion methods

As shown, the highest number of proteins was identified upon applying a combined digestion approach and using GluC and trypsin. However, the choice of proteases used also depends on analytical problem to be addressed as, e.g. for detection of glycosylated proteins, which might

Upon tryptic digestion of proteins, resulting peptides are being separated on a chromatographic column prior to mass spectrometric detection and subsequent bioinformatics analysis. Separation of peptides is being performed either using one-dimensional approach or the multidimensional separation by combining two or more separation technologies prior to MS detection and analysis. For the one-dimensional chromatographic separation approach, peptides are being injected onto the reversed phase nano HPLC column where they are separated according to their hydrophobic interaction with the stationary phase [60]. Figure 2 shows an exemplary base peak chromatogram (BPC) for one-dimensional analysis separation of salivary peptides. Usually, long separation gradients are selected for one-dimensional separation in order to provide the best possible

Figure 3. Base Peak Chromatogram (BPC) of GluC-tryptically generated peptides, which were separated on a reversed

for salivary samples obtained from healthy donors is shown in Figure 2.

4. Chromatographic separation of digested proteins

conditions for peptide separations and large number of identifications.

require additional proteases to be applied.

72 Salivary Glands - New Approaches in Diagnostics and Treatment

phase nano HPLC column.

The use of multidimensional separation methods will increase the number of peptides detected and the number of identified proteins, and, in addition, protein's sequence coverage will be

Figure 4. A number of combinations of different techniques can be used for separation of peptides and proteins in a proteomics approach.

Figure 5. Two-dimensional separation approach for salivary peptides enables identification of higher number of proteins. Data courtesy of Zofia Świątczak (Master Thesis).

improved for identified proteins. Improved sequence coverage for identified proteins is one of the major challenges for proteomics analysis with high importance since it enhances chances for detection of posttranslational modifications (PTM) such as phosphorylation, glycosylation, methylation, etc., which are important as drug targets.

Different types of separation approaches can be used for the two-dimensional approach: strong cation exchange columns, weak anion exchange columns, reversed phase columns at high pH, and other combinations are possible [61–63]. A schematic of possible combinations of chromatographic approaches is shown in Figure 4.

Generally, the use of multidimensional separation will result in increased number of identifications, and Figure 5 shows the comparison of the number of detected proteins upon applying the two-dimensional chromatographic separation with strong cation exchange column used for the first separation dimension.
