**2.2 Proteomics sample preparation**

Trypsin for protein digestion was purchased from Promega Inc. (Vienna, Austria). Solvents for HPLC—methanol (MeOH), acetonitrile (AcN), 2,2,2-trifluoroethanol (TFE), formic acid (FA), heptafluorobutyric acid (HFBA), iodoacetamide (IAA), triethylammonium bicarbonate (TEAB), and dithiothreitol—were purchased from Sigma-Aldrich (Vienna, Austria).

One of the most important steps when analyzing embryo cultivation media is the depletion of serum albumin present in these samples. Tarasova et al. [1] described the innovative method of using immobilized antihuman albumin antibodies for depletion of small sample volumes. Briefly, for depletion of culture media samples from selected embryos, the sample was diluted using the phosphate buffered saline buffer (pH 7.4) consisting of 0.01 M phosphate buffer, 0.0027 M KCl, and 0.14 M NaCl. This buffer was further also used as a washing buffer A upon the sample loading. In order to ensure full sample recovery from the depletion column, a ready-to-use elution buffer from Agilent (pH 2.25) (Agilent Technologies, CA, USA) was used as buffer B. We have developed a new column for the depletion of human albumin by immobilizing the antihuman albumin antibodies to the monolithic support disk, the CIMac-HSA column, especially for analysis of small sample amounts, which also occur in IVF samples. This column was used in an ICS-5000 inert HPLC system (Dionex-Thermo Scientific, Germering, Germany) for albumin depletion with a column flow rate of 0.3 mL/min. Upon sample injection, the loading and washing buffer A was pumped through the column for 5 min, and the flow-through fraction was collected (V = 350 μL). This fractions' volume corresponds to the full absorbance peak and contains all proteins that were not trapped on the column. The albumin was trapped by the interaction with the antibodies on the column's surface, and it was eluted by increasing the amount of the eluting mobile phase from 5 to 10 min. The column was, finally, flushed with the loading buffer A for additional 4 min, and this step was followed by an additional washing step with buffer B and, finally, equilibrating step using, again, the loading buffer A for 13 min. The total time for completing this depletion protocol is 30 min when applying the column flow rate of 0.3 mL/min. During this time, the very important column wash step and the complete re-equilibration of the column preceding the next depletion run is being perfromed. The flow rate used was selected for maximizing the protein's interaction time and was a compromise between the speed and efficacy of operation. If desired, higher column flow rates can be used without losing much of the column's performance [2], but this shall be carefully examined and optimized.

Proteins in both collected fractions and non-depleted samples were depleted using a standard protocol with trypsin. Due to a high concentration of phosphate buffer in collected fractions, 1 M triethylammonium bicarbonate (TEAB) was added to the fractions in order to reach a final concentration of 50 mM TEAB and to dilute the phosphate buffer. For protein denaturation, 10 μL of 0.1% (w/v) Rapigest in 50 m MTEAB were added to all fractions, and reduction of disulfide bonds was performed using dithiothreitol (final concentration of 5 mM DTT) and incubating the reaction mixture for 30 min at 60°C.

For a successful trypsinization, alkylation was performed using iodoacetamide at a final concentration of 15 mM IAA. Upon addition of IAA, the sample was incubated at room temperature, in the dark, for 30 min. An excess of iodoacetamide is inhibiting the trypsin action and needs to be neutralized, which was achieved by adding 2 μL of 50 mM DTT in 50 mM TEAB, vigorous mixing for 2 min at room temperature. Finally, 10 μL of 0.2 μg/μL trypsin solution were added, and samples were incubated for 16 h at 37°C in an incubation oven. Tryptic activity was stopped by acidifying the solution with 1% TFA solution.

For LC–MS/MS analysis, 20 μL of the digested and diluted sample (aqueous 0.1% TFA at a ratio of 2:3 (v/v)) were injected for LC–MS/MS analysis.

### **2.3 Chromatographic separation and mass spectrometry detection**

A Dionex UltiMate 3000 RSLC nanoLC system (Dionex-Thermo Scientific) was used for the nanoHPLC separation of tryptic peptides. Mobile phases used for chromatographic separation of tryptic peptides were as follows: (A) 5% of acetonitrile in aqueous 0.1% formic acid and (B) methanol, trifluoroethanol, water and acetonitrile (30:10:10:50 (v/v/v/v) and 0.1% of formic acid). The sample was loaded onto the trap column for washing the residula salts and focusing of the analytes as a small sample front by using loading mobile phase consisting of 0.1% aqueous TFA chilled to 3°C.

The nonlinear gradient of 75 min total running time was used for HPLC separations of tryptic peptides. The gradient was composed using sequential linear steps:

1. 1.0% B min<sup>−</sup><sup>1</sup> for 7 min.

2.0.5% B min<sup>−</sup><sup>1</sup> gradient for 38 min.

3. 1.5% B min − 1 for 20 min.

4. 3.0% B min<sup>−</sup><sup>1</sup> for 10 min.

The HPLC flow was introduced into the maXis Impact UHR TOF mass spectrometer (Bruker Daltonics, Bremen, Germany), and the LC–MS/MS analysis was performed in triplicate for each sample. For peptide fragmentation (MS/MS), collision-induced dissociation was used, and all MS/MS data were acquired in a 300–2000 m/z range using a three-second cycle without further optimization of the instrument's parameters. In order to prevent unnecessary fragmentation of ions with low intensity, the lower threshold for precursor ions was set to 1000 counts. Due to tailing of some peptides and frequently observed co-elution of peaks, a dynamic exclusion was employed, and the duration was set to 120 s. In case where the precursor ion showed higher intensity after the exclusion period of 120 s, it was reconsidered for an additional MS/MS event.

Database search for protein identification was performed using the all-taxonomy Swiss-Prot database (http://www.uniprot.org). For this search, X! Tandem [3] with following parameters was used:

**137**

*Proteomics as a Future Tool for Improving IVF Outcome DOI: http://dx.doi.org/10.5772/intechopen.89880*

1.Precursor mass tolerance of ±10 ppm.

2.Fragment mass tolerance of ±0.3 Da.

**2.4 Data analysis**

index (SIn) (Griffin et al.) [6].

**3. Results and discussion**

**3.1 Albumin depletion in IVF cultivation medium**

3.Maximum of two missed cleavages were allowed.

Some posttranslational modifications of peptides due to the sample preparation and due to biological processes can be expected. For the experiment described, cysteine carbamidomethylation was selected as the fixed modification and methionine oxidation; phosphorylation of serine, threonine, and tyrosine residues, as well as the acetylation of lysine and N-terminal residues, were selected as variable modifications.

All data analysis for the experiments described in this manuscript was analyzed using the method described by Tarasova et al. [1]. The use of pyteomics.pepxmltk converter (https://pypi.python.org/pypi/pyteomics.pepxmltk) and search was described elsewhere [4]. Briefly, this platform was used to convert X! Tandem files to the standard pep.xml format and perform data analysis. All identifications, upon database search, were filtered to meet the requirement of the 1.0% FDR at peptide level. Search results were submitted for a post-search validation using the MPscore software, described earlier [5]. Quantitative information on identified proteins is a substantial requirement for determining the differences between biological samples, and all proteins fulfilling the identification and validation requirements were quantified using the label-free quantitation approach called normalized spectral

The presence of albumin in cultivating medium is necessary for the normal development of embryos. However, its presence is a significant burden for proteomics analysis, and it must be removed prior to further analysis steps. The removal of albumin has been extensively discussed and described in a number of publications [1, 2, 7–9]. Different groups have used a number of methods such as immunodepleting chromatography, molecular weight cutoff filters, peptide libraries, size exclusion chromatography, etc. All these methods have some advantages but also show disadvantages. In case of depleting the albumin from IVF cultivating medium, the very low sample volume (max. of 40 μl) must be taken into consideration. Furthermore, depletion method must be performed fast and must be reproducible over a large number of samples, if intended to be used for fast analysis of clinical samples that shall help making the decision on which fertilized oocyte shall be transferred first and which ones shall be frozen for later procedures. The use of a novel immunoaffinity-based convective interaction media analytical columns (CIMac) for depletion of HSA (CIMac-HSA) was performed in this study, and it proved that it can be used for fast and reproducible albumin depletion from minute sample amounts. The column's architecture and the convective flow-through columns' channels enable a flow rate-independent binding capacity and excellent chromatographic resolution. These characteristics give CIMac-αHSA column some important analytical benefits like shorten time of analysis in comparison to common chromatographic depletion of albumin using silica-based columns, which


Some posttranslational modifications of peptides due to the sample preparation and due to biological processes can be expected. For the experiment described, cysteine carbamidomethylation was selected as the fixed modification and methionine oxidation; phosphorylation of serine, threonine, and tyrosine residues, as well as the acetylation of lysine and N-terminal residues, were selected as variable modifications.

## **2.4 Data analysis**

*Innovations in Assisted Reproduction Technology*

the reaction mixture for 30 min at 60°C.

by acidifying the solution with 1% TFA solution.

for 7 min.

for 10 min.

reconsidered for an additional MS/MS event.

following parameters was used:

gradient for 38 min.

Proteins in both collected fractions and non-depleted samples were depleted using a standard protocol with trypsin. Due to a high concentration of phosphate buffer in collected fractions, 1 M triethylammonium bicarbonate (TEAB) was added to the fractions in order to reach a final concentration of 50 mM TEAB and to dilute the phosphate buffer. For protein denaturation, 10 μL of 0.1% (w/v) Rapigest in 50 m MTEAB were added to all fractions, and reduction of disulfide bonds was performed using dithiothreitol (final concentration of 5 mM DTT) and incubating

For a successful trypsinization, alkylation was performed using iodoacetamide

For LC–MS/MS analysis, 20 μL of the digested and diluted sample (aqueous

A Dionex UltiMate 3000 RSLC nanoLC system (Dionex-Thermo Scientific) was used for the nanoHPLC separation of tryptic peptides. Mobile phases used for chromatographic separation of tryptic peptides were as follows: (A) 5% of acetonitrile in aqueous 0.1% formic acid and (B) methanol, trifluoroethanol, water and acetonitrile (30:10:10:50 (v/v/v/v) and 0.1% of formic acid). The sample was loaded onto the trap column for washing the residula salts and focusing of the analytes as a small sample front by using loading mobile phase consisting of 0.1% aqueous TFA chilled to 3°C. The nonlinear gradient of 75 min total running time was used for HPLC separations of tryptic peptides. The gradient was composed using sequential linear steps:

The HPLC flow was introduced into the maXis Impact UHR TOF mass spectrometer (Bruker Daltonics, Bremen, Germany), and the LC–MS/MS analysis was performed in triplicate for each sample. For peptide fragmentation (MS/MS), collision-induced dissociation was used, and all MS/MS data were acquired in a 300–2000 m/z range using a three-second cycle without further optimization of the instrument's parameters. In order to prevent unnecessary fragmentation of ions with low intensity, the lower threshold for precursor ions was set to 1000 counts. Due to tailing of some peptides and frequently observed co-elution of peaks, a dynamic exclusion was employed, and the duration was set to 120 s. In case where the precursor ion showed higher intensity after the exclusion period of 120 s, it was

Database search for protein identification was performed using the all-taxonomy Swiss-Prot database (http://www.uniprot.org). For this search, X! Tandem [3] with

0.1% TFA at a ratio of 2:3 (v/v)) were injected for LC–MS/MS analysis.

**2.3 Chromatographic separation and mass spectrometry detection**

at a final concentration of 15 mM IAA. Upon addition of IAA, the sample was incubated at room temperature, in the dark, for 30 min. An excess of iodoacetamide is inhibiting the trypsin action and needs to be neutralized, which was achieved by adding 2 μL of 50 mM DTT in 50 mM TEAB, vigorous mixing for 2 min at room temperature. Finally, 10 μL of 0.2 μg/μL trypsin solution were added, and samples were incubated for 16 h at 37°C in an incubation oven. Tryptic activity was stopped

**136**

1. 1.0% B min<sup>−</sup><sup>1</sup>

2.0.5% B min<sup>−</sup><sup>1</sup>

4. 3.0% B min<sup>−</sup><sup>1</sup>

3. 1.5% B min − 1 for 20 min.

All data analysis for the experiments described in this manuscript was analyzed using the method described by Tarasova et al. [1]. The use of pyteomics.pepxmltk converter (https://pypi.python.org/pypi/pyteomics.pepxmltk) and search was described elsewhere [4]. Briefly, this platform was used to convert X! Tandem files to the standard pep.xml format and perform data analysis. All identifications, upon database search, were filtered to meet the requirement of the 1.0% FDR at peptide level. Search results were submitted for a post-search validation using the MPscore software, described earlier [5]. Quantitative information on identified proteins is a substantial requirement for determining the differences between biological samples, and all proteins fulfilling the identification and validation requirements were quantified using the label-free quantitation approach called normalized spectral index (SIn) (Griffin et al.) [6].
