A novel precursor ion discovery method on a hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer for studying protein phosphorylation

Abstract

A tandem quadrupole time-of-flight (Q-TOF) mass spectrometer has been programmed such that phosphorylated peptides can automatically be discovered and identified in a way similar to that of the use of precursor ion or neutral loss scanning, but without the need to scan the quadrupole mass filter. Instead, the method capitalizes on the innate capability of the Q-TOF to record mass spectra and product ion spectra quickly, with good sensitivity and with good mass accuracy. Alternate mass spectra, with and without fragmentation, are recorded at high and low collision energy with the quadrupole operating in wideband mode. The method of analysis is both compatible with and dependant on liquid chromatography for separation of complex mixtures. The method has been demonstrated by searching for the neutral loss of 98 Da (H 3 PO 4 ) from phosphoserine and phosphothreonine residues, or for the phosphorylated immonium ion at m/z 216 from phosphotyrosine. The method also incorporates acquisition of the product ion spectrum from any candidate precursor ions, thereby allowing confirmation of the neutral loss or product ion and providing additional sequence information to assist identification of the protein and assign the site of phosphorylation. (J Am Soc Mass Spectrom 2002, 13, 792-803)

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

“…One of the benefits using the LC-MS E approach for protein identification [44][45][46] is that for every identified protein, a large number of the best ionized peptides and the corresponding fragment with concentrations >1,000 ppm), medium abundance (yellow, concentrations: 500-1,000 ppm), low abundance (green concentrations: 100-500 ppm) and very low-abundance (gray, under 100 ppm). As shown in Table 1, higher purity samples (>98.8% purity) were obtained for PTG1 expressed from CHO-S cell line regardless of the purification column/protocol used.…”

Multiple neutral loss monitoring (MNM): A multiplexed method for post-translational modification screening

“…This cut off was selected to eliminate the low mass background of the solvent [5,8]. Acceleration voltages of 10,15,20,25, and 30 V were applied from Q0 to Q2 to fragment the transmitted peptides in Q2.…”

“…This neutral loss analysis identifies the presence of a modification as well as the associated precursor ion; however, as this scan function requires that a quadrupole be scanned, the duty cycle is in the range of 0.1%, i.e., if a mass range of 1000 Th is scanned in 1 Th increments, only 1/1000 of the time is spent transmitting the ion to the collision cell. The duty cycle drops even lower if a quadrupole time-of-flight mass spectrometer is used for the identification of neutral losses [5]. This time-consuming process requires at least one analysis per post-translational modification, as one must specifically search for a particular modification from a peptide of some particular charge state.…”

“…This limits the total number of modifications screened in LC-MS/MS experiments, especially considering their inverse relationship with sensitivity in a fixed time frame. As well, due to the poor duty cycle, the acquisition rate must be rather long and, thus, the neutral loss scan (as well as the precursor ion scan) is not routinely used for the analysis of subpicomole quantities of peptides by LC-MS [5]. For these reasons, a novel scan technique has been developed to overcome the limitations of the conventional methods used to monitor for post-translational modifications.…”

Development of new methodologies for the mass spectrometry study of bioorganic macromolecules

“…With its high-resolution capability, the quadrupole time-of-flight instrument can provide an isotope pattern to support the charge-state assignment. It must also be emphasized that, as shown by Bateman et al (2002), tandem quadrupole time-of-flight (Q-TOF) mass spectrometry gives rise to very good results if used in the precursor-ion or neutral-loss scanning mode.…”