Comparison between Enhanced MALDI In-source Decay by Ammonium Persulfate and N- or C-Terminal Derivatization Methods for Detailed Peptide Structure Determination

Abstract

Amino acid sequencing and more detailed structure elucidation analysis of peptides and small proteins is a very difficult task even if state-of-the-art mass spectrometry (MS) is employed. To make this task easier, chemical derivatization methods of the N terminus with 4-sulfophenyl-isothiocyanate (SPITC) or the C terminus with 2-methoxy-4,5-dihydro-1H-imidazole (Lys-tag) can enhance peptide fragmentation or fragment ionizability, via proton mobility/localization mechanisms making tandem MS (MS(2)) spectra more informative and less demanding for structural interpretation. Observed disadvantages related to both derivatization methods are sample- and time-consuming procedures and the increased number of reaction byproducts. A novel, sulfate radical in-source formation method of matrix-assisted laser desorption ionization (MALDI) MS based on chemically enhanced in-source decay (ISD) can be accomplished by simple addition of ammonium persulfate (APS) in the matrix solution. This method enables effective decomposition of peptide ions already in the first stage of MS analysis where a large number of fragment ions are produced. The resultant MALDI-ISD mass spectra (MS after APS → MALDI-ISD MS) are almost equivalent to conventional, collision-induced dissociation (CID) MS(2) spectra. These fragment ions are further subjected to the second stage of the MS, and consequently, MS(3) spectra are produced, which makes the sequence analysis more informative and complete (CID MS(2) is thus equivalent to CID MS(3)). Multiply stage MS after APS addition showed enhanced sensitivity, resolution, and mass accuracy compared to peptide derivatization (SPITC and Lys-tag) or conventional MS and MS(2) analyses and offered more detailed insight into peptide structure.

MALDI-TOF mass spectrometry-based quantification of C-peptide in diabetes patients

“…Derivatizing reagents, such as ammonia solution, ammonium bicarbonate, ammonium sulfate, dansyl chloride and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), are wildly used for labeling amines, amino acids, peptides, proteins and enhancing the MALDI detection sensitivity [11][12][13][14] by improving the ionization efficiency of target peptides. 15 We chose AQC for chemical modification in C-peptide sample preparation prior to the MALDI analysis in our study, based on preliminary data (not shown) and the previous experience of other groups.…”

Enrichment and Quantitative Determination of 5-(Hydroxymethyl)-2′-deoxycytidine, 5-(Formyl)-2′-deoxycytidine, and 5-(Carboxyl)-2′-deoxycytidine in Human Urine of Breast Cancer Patients by Magnetic Hyper-Cross-Linked Microporous Polymers Based on Polyionic Liquid

“…To settle this issue, ionic liquid was proposed as a relatively suitable monomer. Ionic liquids are heterogeneous types of compounds typically formed by a heterocyclic cation based on substituted imidazole or pyridine systems and an inorganic counteranion. − It has good stability, wide liquid temperature range, and excellent hydrophilia, which are widely applied in chromatographic analysis. Our group has developed an ionic liquid assisted method for the detection of biomolecules, providing the basis for the analysis of 5-mdC oxidation products …”

In-Source Decay during Matrix-Assisted Laser Desorption/Ionization Combined with the Collisional Process in an FTICR Mass Spectrometer

“…The N–C α bond cleavage at the N-terminal side of cysteine residues is high compared to other amino acids, , whereas the ISD ions originating from cleavage of N-terminal side of Gly, Val, and Ile are somewhat low. , In contrast, a proline-rich peptide without a basic chemical group undergoes alternative peptide bond cleavage N-terminal to the first proline of the XPP motif . Recently, it was reported that the peptide bond cleavage in MALDI-ISD can be enhanced by addition of ammonium sulfate or ammonium persulfate in the MALDI matrix solution.…”

“…12,22 In contrast, a proline-rich peptide without a basic chemical group undergoes alternative peptide bond cleavage N-terminal to the first proline of the XPP motif. 23 Recently, it was reported that the peptide bond cleavage in MALDI-ISD can be enhanced by addition of ammonium sulfate 24 or ammonium persulfate 25 in the MALDI matrix solution.…”