Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria
Abstract
:1. Introduction
2. Results
2.1. Selection of Specific Peptides for Detection of AprBp and GseBp Proteins
2.2. Calibration Plots for Quantification of Serine Proteases AprBp and GseBp
2.3. Quantification of AprBp and GseBp Serine Proteases by the Selected Peptides
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Bacterial Strains and Growth Conditions
4.3. Sample Preparation
4.4. Assessment of Specific Proteolytic Activity
4.5. SDS-PAGE
4.6. In-Gel Tryptic Digestion and Peptide Extraction
4.7. In Silico Peptide Selection and Skyline Settings
4.8. Quantitative LC–MRM–MS Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
AprBp | Subtilisin-like protease |
DTT | Dithiothreitol |
ELISA | Enzyme-linked immunosorbent assay |
GseBp | Glutamyl endopeptidase |
GRAS | Generally recognized as safe |
IAA | Iodoacetamide |
IPTG | Isopropyl β-galactopyranoside |
iTRAQ | Isobaric tags for relative and absolute quantitation |
LB | Lysogeny broth |
LC–MS | Liquid chromatography-tandem mass spectrometry |
LIKE | LIa-kontrollierte expression |
LPS | Lipopolysaccharide |
MRM | Multiple reaction monitoring |
OD | Optical density |
QQQ | Triple-quadrupole |
Q1 | First quadrupole |
Q2 | Second quadrupole |
Q3 | Third quadrupole |
rcf | Relative centrifugal field |
rpm | Revolutions per minute |
SILAC | Stable isotope labeling by amino acids in cell culture |
SN | Supernatant |
TCA | Trichloroacetic acid |
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Precursor Ion Q1, m/z | Ion Charge | Product Ion Q3, m/z | Peptide | Declustering Potential (V) | Collision Energy (V) | Retention Time (min) |
---|---|---|---|---|---|---|
AprBp | ||||||
521.27 | 802.42 | APAVHAQGYK | 69.1 | 27.6 | 1.03 | |
+2 | 703.35 | |||||
566.29 | ||||||
487.73 | 860.42 * | NAVDTANNR | 66.7 | 26.4 | 0.79 | |
+2 | 690.31 | |||||
575.28 | ||||||
887.93 | 1145.56 | LENTATPLGNSFYYGK | 95.8 | 40.8 | 5.62 | |
+2 | 1048.50 | |||||
935.42 | ||||||
GseBp | ||||||
655.82 | 993.51 * | TDTNIGNTVGYR | 78.9 | 32.5 | 1.39 | |
+2 | 766.38 | |||||
709.36 | ||||||
418.71 | 723.33 | ISGYPGDK | 61.6 | 23.9 | 1.11 | |
+2 | 636.29 | |||||
416.21 | ||||||
722.87 | 1130.58 | ATAAFVEFINYAK | 83.8 | 34.9 | 6.46 | |
+2 | 983.51 | |||||
884.45 |
Strain | Total Protein Concentration (μg mL−1) in SN, Inducer "−" | Total Protein Concentration (μg mL−1) in SN, Inducer "+" | Target Protein Concentration (μg mL−1) in the Vial, Inducer "−"/"+" | Target Protein Concentration (μg mL−1) in SN, Inducer "−"/"+" |
---|---|---|---|---|
Subtilisin-like protease (AprBp) | ||||
B. pumilus 3–19 | 4.5 | – | 6 | 1.63 |
B. subtilis AT1 | 0.32 | 0.33 | 0 | 0 |
B. subtilis MRB044 (SPAprBp) | 2 | 5 | 0.2/5 | 0.024/1.5 |
B. subtilis MRB046 (SPYngk) | 1.3 | 1.8 | 0.45/0.25 | 0.005/0.03 |
Glutamyl endopeptidase (GseBp) | ||||
B. pumilus 3–19 | 4.5 | – | 5 | 1.3 |
B. subtilis AT1 | 0.32 | 0.33 | 0/0 | 0/0 |
B. subtilis MRB047 (SPGseBp) | 1.2 | 1.65 | 0.005/0.05 | 0.00036/0.005 |
B. subtilis MRB049 (SPYngk) | 2.06 | 3.2 | 0.05/0.3 | 0.006/0.06 |
Strain | Relevant Genotype | Source |
---|---|---|
B. pumilus 3–19 | StrR | Laboratory of Biosynthesis and Bioengineering of Enzymes, KFU |
B. subtilis: | ||
BG2036 | ΔaprE-684, ΔnprE522 | (Yang et al [39]) |
AT1 | ΔaprE-684, ΔnprE522, pLIKE-rep | (Tikhonova et al [23]) |
MRB044 | pLIKE-rep + SPAprBp + AprBp | (Tikhonova et al [23]) |
MRB046 | pLIKE-rep + SPYngk + AprBp | (Tikhonova et al [23]) |
MRB047 | pLIKE-rep + SPGseBp + GseBp | (Tikhonova et al [23]) |
MRB049 | pLIKE-rep + SPYngk + GseBp | (Tikhonova et al [23]) |
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Toymentseva, A.A.; Koryagina, A.O.; Laikov, A.V.; Sharipova, M.R. Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria. Int. J. Mol. Sci. 2020, 21, 4924. https://doi.org/10.3390/ijms21144924
Toymentseva AA, Koryagina AO, Laikov AV, Sharipova MR. Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria. International Journal of Molecular Sciences. 2020; 21(14):4924. https://doi.org/10.3390/ijms21144924
Chicago/Turabian StyleToymentseva, Anna A., Anastasia O. Koryagina, Alexander V. Laikov, and Margarita R. Sharipova. 2020. "Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria" International Journal of Molecular Sciences 21, no. 14: 4924. https://doi.org/10.3390/ijms21144924