Fragmentation of multiply-charged intact protein ions using MALDI TOF-TOF mass spectrometry

Abstract

Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach. ragmentation of intact protein gas-phase ions, termed top down proteomics, is becoming more frequently utilized primarily in ion trapping instruments. As was first reported by Loo et al.[1], multiply-charged intact protein ions are formed via electrospray ionization (ESI) and can be collisionally activated to produce sequence-specific fragment ions. Typically, the fragmentation of highly multiply charged intact proteins results in a product ion spectrum displaying a multitude of product ions possessing a range of charge states making spectral interpretation challenging. The high resolving power and mass accuracy of Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry has made this instrument platform the method of choice for the interpretation of the complicated product ion spectra produced by fragmentation of multiply charged protein ions. Indeed, automated interpretation algorithms have been constructed based on this approach [2,3]. Using FTMS, proteins over 200 kDa have been successfully fragmented and sequence-specific product ion assignments have been made [4]. Lower mass resolving quadrupole ion trapping instruments have also been successfully utilized for top down proteomics experiments. To meet the challenge of spectral interpretation ion/ion reactions have been employed to reduce the product ion charge by subjecting the product ion population to proton transfer reactions, thereby resulting in the formation of predominantly singly charged product ions and a more readily interpretable product ion spectrum [5]. This strategy removes the product ion charge-state ambiguities arising from dissociation of large multiply charged protein ions and has been successfully demonstrated on ions as large as elastase (25.9 kDa) although it is limited by the m/z range of the instrument [6]. Matrix-assisted laser desorption ionization tandem time-of-flight (MALDI TOF-TOF) mass spectrometry has also been used to produce product i...

Top-down proteomic identification of bacterial protein biomarkers and toxins using MALDI-TOF-TOF-MS/MS and post-source decay

“…MALDI-TOF-TOF was initially developed for high-throughput tandem mass spectrometry (MS/MS) of peptides of digested proteins (i.e., " bottom-up " proteomics; Washburn et al 2001 , Aebersold andMann 2003 ), but it was quickly demonstrated that small or modest-sized, nondigested protein ions could also be analyzed by MS/MS to allow a topdown proteomic identification (Lin et al 2003, Demirev et al 2005, Liu and Schey 2008, Fagerquist et al 2009. As the ions generated by MALDI are primarily (although not exclusively) singly charged, the fragment ions are also singly charged, which greatly simplifies data interpretation.…”

“…The fragment ions generated may be sufficient for peptide mass fingerprinting, resulting in a searchable sequence " tag " that, when combined with the mass of the protein, can be used to identify the protein from a database. ISD fragment ions can be further mass-selected and fragmented and the fragment ions analyzed, i.e., MS/MS, using either collision-induced dissociation (CID) (Liu and Schey 2008 ) or laser-induced dissociation (LID) (Spengler 1997, Lin et al 2003, Demirev et al 2005, Fagerquist et al 2009. MS/MS of ISD fragment ions provides further sequence coverage extending to the N-or C-termini.…”

“…This approach works most effectively with multiply charged protein ions, e.g., doubly and triply charged protein ions. Liu and Schey (2008) demonstrated top-down identification of pure protein standards by CID of doubly and triply charged ions using α -cyano-4-hydroxycinnamic acid (CHCA) matrix. Because of its higher ionization energy, CHCA is more likely to generate multiply charged protein ions compared with other MALDI matrices (Frankevich et al 2003 ).…”

Possible Evidence of Amide Bond Formation Between Sinapinic Acid and Lysine-Containing Bacterial Proteins by Matrix-Assisted Laser Desorption/Ionization (MALDI) at 355 nm

“…Although initially developed as a high throughput platform for bottom-up proteomics, MALDI-TOF-TOF instruments are increasingly utilized for top-down proteomic identification of non-digested proteins using either in-source decay (ISD) [28][29][30] or post-source decay (PSD) [31][32][33][34][35][36]. As ISD and PSD occur in different regions of the mass spectrometer there are significant differences in ion fragmentation and polypeptide backbone cleavage.…”

Development of a microfluidics‐based gel protein recovery system

“…Top-down approaches are extremely important for the accurate determination of protein molecular weights, which can be used together with partial protein sequences to provide a level of understanding that cannot be achieved with one piece of information alone [6]. Furthermore, several researchers have demonstrated the ability to obtain structural information from fragmentation of intact proteins in gas phase [7][8][9][10]. The main advantage of performing MS/MS on an intact protein in this way, is that, the whole sequence is available for analysis, as well as any associated post-translational modifications [9].…”