Classical method of trypsin digestion in solution
Gel electrophoresis is a widely used method for studying proteins, allowing for the separation of individual protein fractions based on differences in size, charge, or conformation. In the context of this study, gel electrophoresis was performed to analyze the proteins of interest, β-casein (βCN) and β-lactoglobulin (βLG). This technique provided information on the purity of the protein samples and offered a preliminary estimation of their molecular masses. Visual evaluation of the electrophoresis results (qualitative assessment) was conducted, as depicted in Fig. S1. The obtained bands indicated a mass of approximately 23 kDa for βCN and 18 kDa for βLG.
Following gel electrophoresis, protein bands were excised from the polyacrylamide gel, and an in-gel digestion process was carried out. This process included washing the gel pieces, followed by reduction and alkylation steps, in-gel digestion, and subsequent extraction of the resulting peptides, as described in the experimental section. Prior to spectrometric analysis, the peptide samples underwent purification and concentration using ZipTip pipette tips. This step was performed to assess whether this particular sample preparation method could potentially improve the results obtained (2.5. Spectrometric study).
Capillary enzyme reactors (CER) – the efficiency of the μ-IMER produced
Attachment of the enzyme to the support structure is achieved by hydrophobic, electrostatic or covalent reaction interactions. Choosing the correct support can be a real challenge because the enzyme loading, porosity or pH stability must be taken into account. Many factors affect the effectiveness of digestion, these include the choice of support, residence time as well as the dynamics of the flow supporting the digestion. To allow enzyme-substrate interactions, a longer reaction (residence) time is usually required. The residence time needs to be increased if reduced efficiency is observed in IMERs in comparison to the free enzyme, or if reduced sequence coverage is observed by MS, particularly if no change in substrate affinity is observed [10].
The activity (efficiency) of the prepared microreactor was checked on the basis of the trypsin-catalyzed hydrolysis reaction of N-α-benzoyl-arginine ethyl ester (BAEE), as by the action of trypsin, BAEE is hydrolyzed to N-α-benzoyl-arginine (BA) and ethanol. Trypsin was chosen because it cleaves the C-terminal of arginine and lysine, thus leaving positively charged amino acids on the newly formed C-terminal, which promotes ionization and fragmentation [18]. For BAEE, the product (BA) of the reaction was separated from the substrate (BAEE) by capillary liquid chromatography, while the yield of the reaction was evaluated using the peak area of the substrate. Due to the similar absorption of radiation by both the substrate and the product, the detection was carried out at λ = 223 nm. Initially, isocratic elution with 40% acetonitrile (ACN) in the presence of 0.1% trifluoroacetic acid (TFA) was used, but due to the long analysis time, gradient elution was applied. The chromatographic process was carried out from 20% ACN content in the presence of TFA in the mobile phase, increasing it to 40%. TFA is the agent most commonly used to separate peptides and proteins in chromatography and acts as an ion-pairing agent. Fig. 2 shows the effect of substrate concentration (0.5–75 mM) and temperature (23 ºC and 37 ºC) on the efficiency of the prepared microreactor at the applied flow rate F = 1 μL/min.
For solutions with low concentrations of C = 0.5 mM, 1 mM, and 2 mM under T = 23 ºC, no signals from the substrate (BAEE) were recorded, indicating 100% hydrolysis of BAEE. For solutions of higher concentrations (10, 12.5, 15, 20, 25, 35, 50, 62.5, and 75 mM), the degree of hydrolysis showed values ranging from 91.23–27.74%, which is related to the fact that unreacted ester remains in the eluate. Hydrolysis of the ester occurring at a lower rate may also be due to too short a contact time between the enzyme and the substrate. For higher temperature (T = 37 ºC), 100% hydrolysis of BAEE was also observed for solutions with concentrations of C = 0.5 mM, 1 mM, and 2 mM, and for concentrations of 10 mM, 12.5 mM, and 15 mM. For higher concentrations (20–75 mM), the degree of hydrolysis showed values in the range of 98.60–36.24%. In terms of laboratory conditions, it is convenient to conduct the reaction at room temperature (23 ºC), however, the optimal temperature for trypsin activity is 37 ºC [19]. Hence, further studies of the activity of the microreactor against proteins (βCN and βLG) were conducted at 37 ºC.
Capillary enzyme reactors (CER) – protein digestion in μ-IMER
The use of liquid chromatography allowed the separation of milk proteins βCN and βLG. Considering that protein digestion requires longer contact time with trypsin compared to the model BAEE ester, a lower flow rate of F = 0.05 μL/min was used.
The chromatogram (Fig. 3) showed very good quality separation and clear signals from the studied proteins.
The first eluted protein in the mixture was βCN, then βLG. The retention times of the eluted signals coincided with those of βCN and βLG included in the standards. In addition, two signals were observed for βLG, which are derived from its genetic variants. A quick and easy way to prepare the sample for the HPLC procedure was developed. Proteins that passed through a microreactor were subjected to analogous tests. It was observed that the intensity of signals for both βCN and βLG decreased compared to the native form of both proteins and additionally new signals appeared. The results indicate the activity of the microreactor against the proteins studied.
Spectrometric study
Proteomic research heavily relies on mass spectrometry (MS) coupled with various sample preparation techniques and separation methods. However, the complexity of proteomic samples poses significant challenges in developing fast and efficient sample preparation methods for subsequent MS analysis [20]. In our study, we conducted a detailed characterization of the primary structure of two proteins, β-casein (βCN) and β-lactoglobulin (βLG). Through careful analysis, we determined the molecular weights of intact βCN (Fig. 4A) and βLG (Fig. 5A) to be approximately 23,885.90±0.216 Da and 18,307.55±0.145 Da, respectively. Additionally, besides the monomeric forms of the proteins, the presence of dimers, trimers, and in the case of βLG, even tetramers and pentamers, was observed. Interestingly, in the case of βLG, we also observed the presence of dimers [2M-H]+ and various oligomers such as trimers [3M-H]+, tetramers [4M-H]+, and pentamers [5M-H]+ (Fig. 5A). These findings align with the information provided in our previous research [8] provides valuable insights into oligomeric forms study of βLG.
To gain further insights, the proteins underwent extensive analysis with a focus its peptides modifications. Tryptic digestion was performed, and the resulting peptides were subjected to PMF analysis in positive mode (Fig. 4 B, Fig. 5 B). Peptides were identified using BioTools software, employing non-standard cysteine searches modified to account for carbamidomethylation, oxidation, and phosphorylation. Tables 1S and 2S present the individual masses of the detected peptides, their sequences, the sites of oxidation and phosphorylation, and the sequence coverage considering the two different sample preparation methods: with and without the use of ZipTip pipette tips. The phosphorylation was observed specifically on serine residues. The mass spectra of the identified protein peptides are shown in Fig. 4 C, D and Fig. 5 C, D.
The in-depth analysis provided valuable information regarding the peptide sequences, and their modifications, contributing to a comprehensive understanding of the proteins βCN and βLG in this study.
To identify the proteins β-casein (βCN) and β-lactoglobulin (βLG), enzymatic digestion was performed using two different methods: the classical in-solution method and the μ-IMER method. The aim was to determine the sample preparation method that would provide the highest degree of sequence coverage, allowing for more accurate protein identification (Fig. 6). In the classical method, the proteins were digested in solution following the established protocol. For the μ-IMER method, the proteins were subjected to digestion within the flow-through microreactor, which offers advantages such as shorter digestion times and improved mass transfer. Additionally, to assess the impact of ZipTip pipette tips on sequence coverage, some samples underwent peptide enrichment using the chromatographic bed of the tips. By comparing the results obtained from the different digestion methods and sample preparation techniques, the most optimal approach for achieving the highest sequence coverage was determined. This selection is crucial for accurate protein identification, enabling a comprehensive understanding of the βCN and βLG proteins. The evaluation of the various methods and techniques allowed for the identification of the most suitable sample preparation method for subsequent analyses.
The findings reveal that the sequence coverage for the protein β-casein (βCN), when subjected to classical digestion in solution was 20%±2%, indicating that a fifth of the protein's sequence was analyzed and identified. This is specifically referring to the presence of oxidized molecules in the protein's structure. However, when the ZipTip pipette tips were used in conjunction with classical digestion, there was an improvement in the sequence coverage by 6%, increasing it to 26%±1%. These ZipTip pipette tips, therefore, appear to enhance the detection and analysis capabilities of the sequence coverage process. The use of a flow-through microreactor, also known as a μ-IMER, further boosted the sequence coverage, bringing it to 33%±1.5%. This shows that the μ-IMER provides a more efficient environment for protein digestion, leading to an increased understanding of the protein's sequence. This value was further enhanced to 41%±3%, when the ZipTip pipette tips were applied post-digestion in the μ-IMER, meaning the combination of the μ-IMER and ZipTip was particularly effective in improving sequence coverage. This method, in fact, provided double the sequence coverage compared to the classical digestion method alone. The researchers also tested a second protein, β-lactoglobulin (βLG). Here, the classical digestion method offered significantly higher sequence coverage – 49%±2%, without ZipTip and 60%±4%, with ZipTip. Again, using the μ-IMER led to an increase in sequence coverage, in this case to 65%±3%. With the additional use of ZipTip, sequence coverage reached 79%±4%, which is notably higher than the results achieved with the βCN protein. It is crucial to mention that for both proteins, phosphorylations – a crucial post-translational modification in protein function – were identified. However, no phosphorylation was detected in the βCN protein using the classical digestion method without ZipTip. Conversely, when ZipTip was incorporated after classical digestion, the sequence coverage for phosphorylated βCN sequences increased to 27%±4%, and when used with the μ-IMER and μ-IMER-ZipTip, it further increased to 30%±2%, and 33%±1%, respectively. In the case of βLG, the use of ZipTip allowed for the detection of a higher proportion of modified peptides for both classical digestion (79%±2%,) and μ-IMER (79%±4%). Hence, in the context of βCN, the application of ZipTip significantly improved the detection of oxidized molecules and phosphorylation, and similar enhancements were observed for βLG. This indicates that the use of ZipTip, particularly in combination with μ-IMER, provides a more effective method for protein sequence coverage and post-translational modification analysis.
The concept of utilizing ZipTip pipette tips is akin to the traditional solid-phase extraction (SPE) technique. The primary purpose of these methods is to augment the concentration of the analytes (components of interest) within the sample and, if required, to purify the sample by eliminating unwanted substances. Both these processes can enhance sequence coverage, which is achieved by amplifying the number of analyte molecules that undergo the Matrix-Assisted Laser Desorption/Ionization (MALDI) process. Additionally, the removal of potential contaminants contributes to a more accurate and unobstructed analysis. However, it's important to note that while these processes increase the concentration of the analytes, they might also inadvertently preconcentrate unwanted impurities. Hence, one should remain cautious about this possibility during such operations. In the study under discussion, it's clear that the application of ZipTip amplified the degree of sequence coverage in the case of oxidized β-casein (βCN) and β-lactoglobulin (βLG), and also enhanced the detection of phosphorylated βCN in both the classical in-solution digestion and μ-IMER methods. Nevertheless, it is observable that the sequence coverage was superior when the μ-IMER method was used compared to the classical in-solution digestion. In particular, the use of the combined μ-IMER-ZipTips methodology yielded the best results for βCN. These results emphasize the μ-IMER's potential for preparing samples swiftly for MALDI-Time of Flight (TOF) Mass Spectrometry (MS) analysis. This revelation highlights the immense potential of using the μ-IMER method to swiftly prepare samples for MALDI-TOF MS analysis. As such, it's an encouraging prospect for advancing the field of proteomics, which involves the large-scale study of proteins, their structures, and functions. By enhancing the sequence coverage and improving the detection of post-translational modifications like oxidation and phosphorylation, these methods could significantly improve the quality and depth of proteomic studies.
A key type of PTM is phosphorylation, which is essentially the addition of a phosphate group to a protein. Phosphorylations have an instrumental role in a wide range of cellular activities. Moreover, they are linked to the development of various health conditions. For instance, abnormalities in phosphorylation processes are associated with the development of cancer and neurodegenerative disorders. This makes the analysis of phosphorylation and other PTMs an essential component of disease research and potential therapeutic intervention strategies. However, the analysis of PTMs presents several challenges, particularly in the context of Mass Spectrometry (MS) and Tandem Mass Spectrometry (MS/MS), which are widely used analytical techniques in proteomics. Some PTMs are unstable during these analyses and may break down or change, making their identification and characterization difficult. Furthermore, several modifications result in the formation of hydrophilic (water-attracting) products. These products can make the handling and purification of PTM samples prior to MS analysis more complex, as they may not interact favorably with the commonly used techniques that are designed for hydrophobic (water-repelling) molecules. PTMs can also affect the efficiency of proteases, such as trypsin, which are enzymes used to break down proteins into smaller peptides for easier analysis. PTMs can lead to unusual cleavage patterns or larger-than-expected peptide products, complicating the interpretation of results. Additionally, the presence of certain PTMs can reduce the ionization and detection efficiency in MS. Ionization is a crucial step in MS where molecules are charged so they can be moved and measured by an electric or magnetic field. Reduced ionization can lead to lower detection levels, potentially obscuring significant findings. Lastly, when a protein has multiple PTMs, the resulting MS and MS/MS data sets can be incredibly complex and difficult to interpret. Each modification can affect the protein's behavior in the MS, leading to a wide array of potential products and signals to be interpreted [21]. Overall, while the analysis of PTMs can offer critical insights into protein function and disease development, it also presents various challenges that require sophisticated techniques and careful interpretation of the generated data.
In-solution digestion using proteases, which are enzymes that break down proteins into smaller fragments, results in a complex reaction mixture. This mixture contains polypeptide fragments produced not only from the target protein but also from the autolysis, or self-digestion, of the proteases themselves. This additional layer of complexity complicates the composition of the polypeptide mixture and makes subsequent analysis of the Mass Spectrometry (MS) spectra more challenging. Autolysis peaks can suppress peptide signals in the MS spectra, thereby reducing the sensitivity of the method. In simple terms, the self-digested enzyme fragments can interfere with the detection of the actual target protein fragments, making it harder to identify and analyze these components. To address these issues, one strategy is the immobilization of enzymes on solid supports or other carriers. By fixing the enzymes onto a solid structure, autolysis can be minimized while maintaining a high enzyme-to-substrate ratio, which is crucial for efficient protein digestion [22]. A particularly effective method in this regard is the use of a flow-through microreactor, also known as μ-IMER. This technique offers several notable advantages over traditional in-solution digestion. First, it greatly accelerates the digestion process, enabling higher throughput than common protocols used in classical methods, such as those involving gel preparation. Furthermore, the μ-IMER method reduces the complications associated with sample handling and processing. For example, it can minimize concerns related to pipette transfer, which is a common source of error and variability in traditional laboratory protocols [23]. By streamlining and improving the digestion process, the μ-IMER method can enhance the reliability and efficiency of proteomic analyses.