Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells
Abstract
1. Introduction
2. Results and Discussion
2.1. Chemistry
2.2. Mass Spectrometry Analysis
2.2.1. Analysis of Reactive NHS Ester 5a
2.2.2. Analysis of Conjugate 7a, i.e., Peptide Ac-AKF-NH2 Tagged with Diethyl Derivative 5a
2.2.3. Analysis of Conjugate 7b, i.e., Peptide Ac-AKF-NH2 Tagged with Dioctyl Derivative 5b
2.2.4. Analysis of Conjugate 7c, i.e., Peptide Ac-AKF-NH2 Tagged with Pyrrolidine Derivative 5c
2.2.5. Analysis of Conjugate 8, i.e., Tetrapeptide Ac-AAAK Tagged with Diethyl Derivative 5a
2.3. Derivatization of Ubiquitin Hydrolysate with Reagent 5a
2.4. Fluorescence Microscopy
3. Materials and Methods
3.1. General Information
3.2. Chemical Synthesis
3.2.1. Synthesis of 8-Carboxy-5,7-Dimethyl-2,2-Dioctyl-2,3-Dihydro-[1,2,4]Triazolo[4,3-a]Pyridin-2-ium Chloride (3b)
3.2.2. Synthesis of Reactive Safirinium P (5b,c) and Q (6b,c) Probes
3.2.3. Synthesis of Peptide Conjugates 7a-c
3.2.4. Synthesis of the Derivatized Peptide 8
3.3. Tagging the Ubiquitin Hydrolysate with 5a
3.4. Fluorescence Microscopy of Bacteria
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
CID | collision-induced dissociation |
DCM | dichloromethane |
EIC | extracted ion chromatogram |
ESI | electrospray ionization |
FT-ICR | Fourier-Transform Ion-Cyclotrone-Resonance |
DIC | N,N-diisopropylcarbodiimide |
DMF | dimethylformamide |
DHX | deuterium-hydrogen-exchange |
GFP | green fluorescence protein |
HDX | hydrogen-deuterium exchange |
HPLC | high performance liquid chromatography |
ITs | ionization tags |
LB | Luria broth |
LC | liquid chromatography |
MRM | multiple reaction monitoring |
MS | mass spectrometry |
MS/MS | tandem mass spectrometry |
NHS | N-hydroxysuccinimide |
NMR | nuclear magnetic resonance |
OD | optical density |
Oct | octyl |
PAGE | polyacrylamide gel electrophoresis |
PBS | phosphate-buffered saline |
QAS | quaternary ammonium salts |
RP | reverse-phase |
TEA | triethylamine |
TEAB | triethylammonium bicarbonate |
TFA | trifuoroacetic acid |
TIC | total ion chromatogram |
TIS | triisopropylsilane |
TRITC | tetramethylrhodamine-isothiocyanate |
References
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No | Peptide Sequence | [M+H]+ | [M+Saf]1+ | [M+2H]2+ | [M+H+Saf]2+ | [M+2Saf]2+ | [M+3H]3+ | [M+2H+Saf]3+ | [M+H+2Saf]3+ | [M+3Saf]3+ | [M+4H]4+ | [M+4Saf]4+ |
---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | NVKAKIQDKEGIPPDQQRL | - | - | - | - | 726.4061 | - | - | - | 545.0564 | - | |
2 | NVKAKIQDKEGIPPDQQ | - | 954.5129 | - | - | - | - | - | - | - | - | |
3 | NVKAKIQDKEGIPPDQ | - | 890.4837 | - | - | - | - | - | - | - | 676.8827 | |
4 | AGKQLEDGRTLSD | - | 695.3521 | - | 926.4893 | - | 540.9496 | 617.9953 | - | - | ||
5 | VKTLTGKTITL | - | 587.8740 | - | - | - | - | - | 623.3889 | - | - | |
6 | YNIQKESTL | 1095.5681 | - | 548.2877 | - | 779.4249 | - | - | - | 596.9981 | - | - |
7 | YNIQKEST | 982.4840 | - | - | - | 722.8829 | - | - | - | - | - | - |
8 | NIQKESTL | - | - | - | 582.3246 | 697.8932 | - | - | - | - | - | - |
9 | VKTLTGKT | 847.5248 | - | 424.2661 | - | - | - | - | - | 514.3170 | - | - |
10 | AGKQLEDG | 817.4051 | - | - | - | 640.3434 | - | - | - | - | - | - |
11 | AGKQLED | - | - | - | - | 611.8326 | - | - | - | - | - | - |
12 | QRLIF | 676.4141 | - | - | - | - | - | - | - | - | - | - |
13 | EVEPSD | 675.2832 | 906.4204 | - | - | - | - | - | - | - | - | - |
14 | VKTLTG | 618.3821 | - | - | - | 540.8319 | - | - | - | - | - | - |
15 | KESTL | 577.3192 | - | - | - | 520.3004 | - | - | - | - | - | - |
16 | RLRGG | 558.3471 | - | - | - | - | - | - | - | - | - | - |
17 | MQIF | 538.2694 | 769.4066 | - | - | - | - | - | - | - | - | - |
18 | HLVL | 481.3133 | 712.4505 | - | - | 472.2975 | - | - | - | - | - | - |
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Fedorowicz, J.; Wierzbicka, M.; Cebrat, M.; Wiśniewska, P.; Piątek, R.; Zalewska-Piątek, B.; Szewczuk, Z.; Sączewski, J. Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells. Int. J. Mol. Sci. 2020, 21, 9643. https://doi.org/10.3390/ijms21249643
Fedorowicz J, Wierzbicka M, Cebrat M, Wiśniewska P, Piątek R, Zalewska-Piątek B, Szewczuk Z, Sączewski J. Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells. International Journal of Molecular Sciences. 2020; 21(24):9643. https://doi.org/10.3390/ijms21249643
Chicago/Turabian StyleFedorowicz, Joanna, Magdalena Wierzbicka, Marek Cebrat, Paulina Wiśniewska, Rafał Piątek, Beata Zalewska-Piątek, Zbigniew Szewczuk, and Jarosław Sączewski. 2020. "Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells" International Journal of Molecular Sciences 21, no. 24: 9643. https://doi.org/10.3390/ijms21249643
APA StyleFedorowicz, J., Wierzbicka, M., Cebrat, M., Wiśniewska, P., Piątek, R., Zalewska-Piątek, B., Szewczuk, Z., & Sączewski, J. (2020). Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells. International Journal of Molecular Sciences, 21(24), 9643. https://doi.org/10.3390/ijms21249643