Stewart Morley and Dr. John Prince, Department of Chemistry & Biochemistry
Introduction
Proteomics uses highly sensitive mass spectrometers to measure peptide fragment masses from enzymatically digested proteins. Measuring the mass of the peptide and fragments produced in the mass spectrometer allows for identification of its parent protein. Phosphoproteomics analyzes peptides that have been phosphorylated. Phosphoproteomics identifies phosphorylation sites, network relationships between proteins, and how cells respond to changes in their environment. Mass spectrometers typically ionize peptides to carry positive charges. This charge aids to draw molecules into the instrument. Phosphate modifications reduce charge on their molecules, reducing the positive charge on phosphopeptides, creating neutral, or negatively charged species. The lack of charge on these species results in a loss of detection.1-2
The most common approach to fragmenting peptides in mass spectrometers involved a process known as collision induced dissociation (CID). CID uses an inert gas that collides with ionized peptides that increases their vibrational energy causinging them to fragment into smaller pieces. These fragments provide sequence information of the peptide and identifies the parent protein. Proper identification requires fragmentation along the peptide backbone. Detection losses occur when analyzing phosphopeptides because they fragment more readily at the phosphoester bond instead of the peptide bacbone.3 Figure 1 illustrates improper phosphopeptide fragmentation. Svane et al recently published a method of phosphate analysis that overcomes these difficulties.4 Continuing their work, we explored the use of a large organic molecule that binds to phosphate modifications to phosphopeptides, decreasing their negative charge and stabilizing the phosphoester bond.
Materials and Methods
Synthesis of the protecting group molecule was performed according to the procedure outlined by Ghiladi et al.5 Synthesized [Gabpbp(O)(OH)]2+ was then reacted with phosphopeptide standards, singly digested protein, and digested complex protein samples in an approximate ratio of 0.5:5 μM peptide to [Gabpbp(O)(OH)]2+. The large volume of the reaction solution was concentrated using a centrifugal evaporator to approximately 30 uL. Mass spectrometry on all treated samples was performed using a Thermo Fisher LTQ orbitrap mass spectrometer. Data analysis was performed using Mascot database search engine.
Results
We successfully synthesized Hbpbp and [Gabpbp(O)(OH)]2+ and confirmed their presence by NMR and ESI-MS. We also performed NMR and ESI-MS on a six-month-old sample of [Gabpbp(O)(OH)]2+ stored at 4°C to examine the molecules stability over time. The experiments on the six month old sample indicated that [Gabpbp(O)(OH)]2+ still existed in high abundance and had not degraded.
A [Gabpbp(O)(OH)]2+ modification was created within the Mascot search engine with monoisotopic mass value of 804.123, which was then used to search for phosphopeptides that had formed a complex with [Gabpbp(O)(OH)]2+. Phosphopeptide standards, single phosphoproteins, and complex solutions of peptides treated with [Gabpbp(O)(OH)]2+ did not return any results indicating that complexation had occurred.
Discussion
Consistent phosphoproteomic results were not obtained using the synthesized [Gabpbp(O)(OH)]2+ dimetal molecule. The results were puzzling since we were able to reproduce the same conditions reported by Svane et al yet we did not obtain any positive identification of a phosphopeptide associated with [Gabpbp(O)(OH)]2+. When we compared our results to the data reported by Svane et al we noticed a few peculiarities. One of these peculiarities was that the spectra produced by Svane et al lack noise at low m/z values. This is curious because every spectrum will possess these low m/z values due to atmospheric contaminants.
We also suspected that perhaps the synthesized molecule could not be stored for long periods of time without degrading. To test whether [Gabpbp(O)(OH)]2+ was still intact we performed ESIMS and NMR analysis on a sample nearly six months old. The experiments indicated that the molecule remained intact and its degradation should not have affected its application. This leads us to think that that association between [Gabpbp(O)(OH)]2+ and gaseous phosphopeptides is unstable. If true, dimetal protection may solve the instability of phosphoester bonds but also creates an unstable, non-covalent association between [Gabpbp(O)(OH)]2+ and phosphopeptides.
Although success may be obtained when analyzing simple samples, widespread use of this method will only occur if it can be applied towards complex samples.
References
- Lehmann, W. D.; Kruger, R.; Salek, M.; Hung, C. W.; Wolschin, F.; Weckwerth, W., Neutral loss-based phosphopeptide recognition: A collection of caveats. J Proteome Res 2007, 6 (7), 2866-2873.
- Salek, M.; Lehmann, W. D., Neutral loss of amino acid residues from protonated peptides in collision-induced dissociation generates N- or C-terminal sequence ladders. J Mass Spectrom 2003, 38 (11), 1143-1149.
- Rozman, M., Modelling of the gas-phase phosphate group loss and rearrangement in phosphorylated peptides. J Mass Spectrom 2011, 46 (9), 949-955.
- Svane, S.; Kryuchkov, F.; Lennartson, A.; McKenzie, C. J.; Kjeldsen, F., Overcoming the Instability of Gaseous Peptide Phosphate Ester Groups by Dimetal Protection. Angew Chem Int Edit 2012, 51 (13), 3216-3219.
- Ghiladi, M.; McKenzie, C. J.; Meier, A.; Powell, A. K.; Ulstrup, J.; Wocadlo, S., Dinuclear iron(III)-metal(II) complexes as structural core models for purple acid phosphatases. J Chem Soc Dalton 1997, (21), 4011-4018.