3. Sample preparation: depletion of abundant proteins and enzymatic digest

The enzymatic digest of salivary proteins does not differ from digestion of other proteins. Usually, trypsin is being used for proteolytic cleavage of proteins due to its relatively high specificity, availability, and ease of use. Furthermore, tryptic peptides are ideally suited for reversed phase HPLC separation and positive ionization using electrospray MS (ESI). Tryptic peptides are also ideal for using multidimensional separation approaches such as strong cation exchange (SCX), hydrophilic interaction liquid chromatography (HILIC), or electrostatic repulsion interaction chromatography (ERLIC) since they bear positive charges on N-terminus or Lys-residues.

Unlike serum or plasma, saliva does not contain large range and amounts of high abundant proteins such as serum albumin or hemoglobin that can affect sensitivity and selectivity of detection. However, amylase and serum albumin are still proteins with the highest abundance in saliva and can also affect the detection of other low abundant species and should be removed from the sample [57, 58]. In human saliva, alpha-amylase makes about 60% of the abundance of all proteins present, and its removal will help identifying proteins of lower abundance such as cytokines, which can be used as putative biomarkers for different processes. Deutsch et al. [57] have shown a simple yet very effective method for removing alphaamylase and gaining a deeper insight into saliva's proteome. The use of a simple potato starch resulted in sixfold reduction of the amount of alpha-amylase in the sample. Albumin removal can be facilitated by using a number of columns developed and based on immunoaffinity

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Figure 2. Comparison of the number of identified proteins from human saliva by applying different proteases. Courtesy

reactions [59].

of Zofia Świątczak (Master Thesis).

Beside trypsin, other proteases can also be used alone or in combination with trypsin. The aim is either to achieve specific cleavage of proteins for special questions or to achieve smaller peptides and enhance their ionization, detection, and ultimately better sequence coverage for identified proteins. Mostly, proteins such as Lys-C and GluC are applied for pre-digestion before trypsin addition.

Protein digest begins with protein precipitation from saliva. A number of methods have been developed for protein precipitation such as: (1) alcoholic; (2) salting out; (3) applying strong acids (trichloroacetic acid); (4) using acetone; (5) using acetonitrile, etc. Protein precipitation shall help for removing DNA and RNA and their fragments from the sample and for removing lipids.

The Proteomics Core Facility of the Medical University in Vienna applies a modified Wessel-Fluegge [53] method for protein precipitation [54]. Figure 1 shows the sample collection steps prior to protein reduction, alkylation, and addition of the protease.

In addition to trypsin, the use of proteases, e.g. LysC, will help generating smaller peptides prior to separation and detection [55, 56].

Figure 1. Sample collection steps prior to protein reduction, alkylation, and addition of the protease as performed at the Proteomics Core Facility of the Medical University of Vienna (https://www.sarstedt.com/en/products/diagnostic/salivasputum/ product/51.1534.500/).

Unlike serum or plasma, saliva does not contain large range and amounts of high abundant proteins such as serum albumin or hemoglobin that can affect sensitivity and selectivity of detection. However, amylase and serum albumin are still proteins with the highest abundance in saliva and can also affect the detection of other low abundant species and should be removed from the sample [57, 58]. In human saliva, alpha-amylase makes about 60% of the abundance of all proteins present, and its removal will help identifying proteins of lower abundance such as cytokines, which can be used as putative biomarkers for different processes. Deutsch et al. [57] have shown a simple yet very effective method for removing alphaamylase and gaining a deeper insight into saliva's proteome. The use of a simple potato starch resulted in sixfold reduction of the amount of alpha-amylase in the sample. Albumin removal can be facilitated by using a number of columns developed and based on immunoaffinity reactions [59].

3. Sample preparation: depletion of abundant proteins and

The enzymatic digest of salivary proteins does not differ from digestion of other proteins. Usually, trypsin is being used for proteolytic cleavage of proteins due to its relatively high specificity, availability, and ease of use. Furthermore, tryptic peptides are ideally suited for reversed phase HPLC separation and positive ionization using electrospray MS (ESI). Tryptic peptides are also ideal for using multidimensional separation approaches such as strong cation exchange (SCX), hydrophilic interaction liquid chromatography (HILIC), or electrostatic repulsion interaction chromatography (ERLIC) since they bear positive charges on N-terminus or Lys-residues.

Beside trypsin, other proteases can also be used alone or in combination with trypsin. The aim is either to achieve specific cleavage of proteins for special questions or to achieve smaller peptides and enhance their ionization, detection, and ultimately better sequence coverage for identified proteins. Mostly, proteins such as Lys-C and GluC are applied for pre-digestion

Protein digest begins with protein precipitation from saliva. A number of methods have been developed for protein precipitation such as: (1) alcoholic; (2) salting out; (3) applying strong acids (trichloroacetic acid); (4) using acetone; (5) using acetonitrile, etc. Protein precipitation shall help for removing DNA and RNA and their fragments from the sample and for removing lipids.

The Proteomics Core Facility of the Medical University in Vienna applies a modified Wessel-Fluegge [53] method for protein precipitation [54]. Figure 1 shows the sample collection steps

In addition to trypsin, the use of proteases, e.g. LysC, will help generating smaller peptides

Figure 1. Sample collection steps prior to protein reduction, alkylation, and addition of the protease as performed at the Proteomics Core Facility of the Medical University of Vienna (https://www.sarstedt.com/en/products/diagnostic/salivasputum/

prior to protein reduction, alkylation, and addition of the protease.

prior to separation and detection [55, 56].

enzymatic digest

70 Salivary Glands - New Approaches in Diagnostics and Treatment

before trypsin addition.

product/51.1534.500/).

Figure 2. Comparison of the number of identified proteins from human saliva by applying different proteases. Courtesy of Zofia Świątczak (Master Thesis).

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

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

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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

Figure 5. Two-dimensional separation approach for salivary peptides enables identification of higher number of proteins.

performing this kind of analysis.

proteomics approach.

Data courtesy of Zofia Świątczak (Master Thesis).

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 require additional proteases to be applied.
