TEMPO-Assisted Free Radical-Initiated Peptide Sequencing Mass Spectrometry (FRIPS MS) in Q-TOF and Orbitrap Mass Spectrometers: Single-Step Peptide Backbone Dissociations in Positive Ion Mode

Abstract

The present study demonstrates that one-step peptide backbone fragmentations can be achieved using the TEMPO [2-(2,2,6,6-tetramethyl piperidine-1-oxyl)]-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry in a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer and a Q-Exactive Orbitrap instrument in positive ion mode, in contrast to two-step peptide fragmentation in an ion-trap mass spectrometer (reference Anal. Chem. 85, 7044-7051 (30)). In the hybrid Q-TOF and Q-Exactive instruments, higher collisional energies can be applied to the target peptides, compared with the low collisional energies applied by the ion-trap instrument. The higher energy deposition and the additional multiple collisions in the collision cell in both instruments appear to result in one-step peptide backbone dissociations in positive ion mode. This new finding clearly demonstrates that the TEMPO-assisted FRIPS approach is a very useful tool in peptide mass spectrometry research. Graphical Abstract ᅟ.

Collision energies on QTof and Orbitrap instruments: How to make proteomics measurements comparable?

“…Orbitraps were found to be comparable or even outperform QTof in the analytical characterization of small molecules in various contexts (metabolomics, drugs, etc. ), 14–20 as well as in the identification and quantification of proteins using both bottom‐up and intact methods 21–25 . Studies of peptide fragmentation in HCD cells, the appropriate choice of experimental parameters used in proteomics, and studies on the transferability of results and experimental setup between the two instruments are all of significant importance.…”

Development of Novel Free Radical Initiated Peptide Sequencing Reagent: Application to Identification and Characterization of Peptides by Mass Spectrometry

“…including proteins, [1][2][3][4][5][6][7][8][9][10][11] glycans, [12][13][14][15][16][17][18][19][20][21] lipids, [22][23][24][25] and nucleic acids [26][27] . A free radical is generated by three major methods, collision-induced dissociation (CID) of fragile bonds, photodissociation (PD) of fragile bonds, and electron activated dissociation (ExD including ECD and ETD).…”

“…As a CIDbased technique, free radical-initiated peptide sequencing mass spectrometry (FRIPS-MS) has made a significant advancement and has recently gained popularity in the field of proteomics. [1][2][3][4][5][6][7] FRIPS-MS relies on radical-induced dissociation initiated by the generation of hydrogen-deficient radicals, which is generated mainly via homolytic dissociation of fragile bonds by either photoactivation or collision induced dissociation (CID). 5,[7][8] In 2004, Porter's group first demonstrated that free radical-induced peptide cleavage can be initiated by collisional activation of the weak peroxide bond that was added to the peptide via sitespecific modification of the ε-amino group of lysine residues or the N-terminus.…”

“…[1][2][3][4][5][6][7] FRIPS-MS relies on radical-induced dissociation initiated by the generation of hydrogen-deficient radicals, which is generated mainly via homolytic dissociation of fragile bonds by either photoactivation or collision induced dissociation (CID). 5,[7][8] In 2004, Porter's group first demonstrated that free radical-induced peptide cleavage can be initiated by collisional activation of the weak peroxide bond that was added to the peptide via sitespecific modification of the ε-amino group of lysine residues or the N-terminus. 9 Two consecutive applications of CID are needed to achieve the free radical-induced peptide cleavages: the nascent aminyl radical is generated by the first collision activation of peptidemetal complexes, wherein fragmentation of the weak O-O bond is followed by loss of CO 2 ; then fragmentation of the peptide is generated by the aminyl radical upon the second collision activation.…”

“…[28][29][30][31] Later, 2 nd generation FRIPS reagents, employing 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO, a stable free radical) as the free radical precursor, were developed for peptide characterization (Scheme 1). [2][3][4][5]7 Recently, FRIPS has been combined with chemical crosslinking/MS as a powerful tool to elucidate structures of peptides. [32][33][34][35][36] Sinz and Schaefer incorporated either Azo or TEMPO to a cross-linker as the free radical precursor for structure analysis of peptides and proteins.…”