Facile Determination of Phosphorylation Sites in Peptides Using Two-Dimensional Mass Spectrometry

Abstract

Detection and characterization of phosphopeptides by infrared multiphoton dissociation two-dimensional mass spectrometry (IRMPD 2DMS) is shown to be particularly effective. A mixture of phosphopeptides was analyzed by 2DMS without any prior separation. 2DMS enables the data independent analysis of the mixture and the correlation of the fragments to their precursor ions. The extraction of neutral loss lines corresponding to the loss of phosphate under IRMPD fragmentation allows the selective identification of phosphopeptides. Resonance of the 10.6 μm infrared radiation with the vibrational modes of the phosphate functional group produced efficient absorption and high cleavage coverage of the phosphopeptides at much lower irradiation fluence than for nonphosphorylated peptides improving discrimination. Additionally, the localization of the phosphate group was determined.

Narrowband Modulation Two-Dimensional Mass Spectrometry and Label-Free Relative Quantification of Histone Peptides

“…2D MS can be performed on Fourier transform ion cyclotron resonance (FT-ICR) or linear ion trap mass spectrometers and has been successfully applied to the analysis of small molecules, polymers, and in bottom-up and top-down proteomics. − In most studies using 2D MS, the analytes were within an m / z range of several hundred units. In the case of modified histones, the m / z range is typically less than 10 units depending on the charge state of the protein.…”

Phase Correction for Absorption Mode Two-Dimensional Mass Spectrometry

“…Multiple studies have shown that, in the vertical dimension, for fragmentation methods that have a maximum efficiency at the center of the ICR cell, the phase φ f,v of the fragment ion signal is shifted by π [ 23 , 24 , 38 ]. The phase of the fragment ion signal can therefore be expressed as: in which f 1 is the cyclotron frequency of the precursor ion, t 1 is the encoding delay, f min is the lowest frequency in the pulse, and T 1 is the duration of the second pulse.…”

“…Multiple studies have shown that, in the vertical dimension, for fragmentation methods that have a maximum efficiency at the center of the ICR cell, the phase ϕ f,v of the fragment ion signal is shifted by π [23,24,38]. The phase of the fragment ion signal can therefore be expressed as:…”

“…The pulse sequence and data processing have been optimized for analytical use [ 12 , 13 , 14 , 15 ]. 2D FT-ICR MS has been used for applications ranging from small molecules and agrochemicals, to polymers, bottom-up and top-down proteomics [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. 2D MS has also been adapted for high-resolution analysis of precursor ions over narrower mass-to-charge ratio ( m/z ) ranges [ 25 ].…”

Phase Correction for Absorption Mode Two-dimensional Mass Spectrometry

“…The pulse sequence and data processing have been optimized for analytical use [12][13][14][15]. 2D FT-ICR MS has been used for applications ranging from small molecules and agrochemicals, to polymers, bottom-up and top-down proteomics [16][17][18][19][20][21][22][23][24]. 2D MS has also been adapted for high-resolution analysis of precursor ions over narrower mass-to-charge ratio (m/z) ranges [25].…”