Fundamentals and Applications of Tandem Mass Spectrometry in Top-down and Bottom-up Protein Analysis

Owing to the development of electrospray ionization (ESI), various ion dissociation techniques and software algorithms, mass spectrometry has become an indispensable tool for protein analysis. However, new approaches are needed to overcome some of the challenges in protein sequencing, including in the identification of post-translational modifications (PTMs), and in the analysis of protein complexes and protein-protein interaction networks. For PTM identification, commonly used database search algorithms for bottom-up proteomics often fail to identify peptides with unexpected PTMs due to their relatively low abundances and the absence of such modifications in the PTM databases. However, such unexpected PTMs can have important roles in biological functions. Here, we report a novel mass spectrometry method to identify the proteins targets of organophosphate (OP) insecticides in a non-targeted fashion. This method integrates a high-resolution twin-ion metabolite extraction program with Mascot database searching. Using this method, transmethylation was identified as a new reaction pathway for OP insecticides, in addition to the well-known phosphorylation modification that causes acute toxicity. Our results show that this method can be used for the reliable identification of unknown PTMs in complicated biomatrices which may ultimately benefit the discovery of protein biomarkers for a variety of conditions. For protein sequence identification, collision induced dissociation (CID) is the most widely used dissociation technique. In the CID of intact proteins, the fragmentation patterns of the protein ion depend strongly on the protein charge state. An optimal charge state can generate selective fragmentation resulting in a limited number of sequence ions in high abundances which can be useful for protein identification with high sensitivity. However, an accurate model to predict the fragmentation patterns of particular charge states is needed. Here, we report an approach to predict the specific cleavage sites of intact protein ions upon CID by use of an improved electrostatic model for calculating the proton configurations of protein ions at different charge states. The origin of highly selective cleavage sites in the CID of highly charged proteins ions is investigated using an improved electrostatic model, molecular dynamics and hybrid ONIOM simulations. The ONIOM results indicate that the protons located at low-basicity amino acid residues can dramatically reduce the reaction barrier to the cleavages at such amide bonds. The results from our electrostatic model suggest that unlike peptide ions at relatively high charge states, protons at low-basicity amino acid residue sites are electrostatically confined within a relatively narrow range of amino acid residues. Such confined protons can ‘trigger’ the fragmentation at the specific peptide bonds. Our model can potentially be used to predict the specific charge states that yield either specific sequence ions in high abundances, or fragment extensively for optimal protein sequence coverage. Recently, higher energy collision induced dissociation (HCD) has become available for the LTQ Orbitrap. There are some reports in the literature that demonstrate that HCD benefits the de novo sequencing of proteins and the identification of PTM sites. However, studies investigating the potential use of HCD for intact protein sequencing are relatively rare. Here, we systemically compared the performance of HCD and CID for intact protein analysis. Our results indicate that HCD yields significant performance gains compared to CID for obtaining high sequence coverage at relatively low charge states owing to higher ion trapping efficiencies and higher ion collision energies. The origin of the highly specific cleavages in the HCD of highly charged protein ions was investigated. We found that for HCD, highly specific fragmentation sites occur near the first sites that low-basicity amino acids are predicted to be protonated with increasing charge states, which is consistent with the mechanism for the formation of highly specific fragmentation of protein ions in CID. This result provides additional evidence that the fragmentation patterns of highly charged protein ions can be predicted to improve protein identification. Solution-phase labelling experiments have been increasingly used in combination with top-down proteomics to rapidly obtain structural information. However, the low charge state of protein ions formed from native solutions usually result in low sequence coverage which limits spatial resolution. Here, we demonstrate that theta-capillary nanoelectrospray ionization can be used to form protein ions with the highest known charge densities to date from native-like solutions by use of alkyl cyclic carbonate “supercharging” additives. It is anticipated this approach will be particularly promising for top-down hydrogen-deuterium exchange mass spectrometry to obtain significantly more protein structural information in native solutions than by use of more conventional approaches