Charge derivatization of peptides for analysis by mass spectrometry

In vacuo trimethylation of the N-terminus of a lyophilized peptide with methyl iodide was previously reported to enhance the peptide's signal in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and to suppress alkali adduct formation in electrospray ionization mass spectrometry (ESI-MS). Both the signal enhancement and alkali adduct suppression observed for methylated peptides are believed to be due to the permanent positive charge on the N-terminus of the peptide resulting from the formation of a quaternary ammonium moiety. The present work evaluates the general utility of the in vacuo methylation procedure for the MS analysis of peptides, and specifically addresses the issue of whether the methylation of nucleophilic sites other than the N-terminal amine affects the MALDI signal of modified peptides. This work establishes that, although certain side-chain modifications are inevitable using present reaction conditions, the derivatization leads to significant MALDI-MS signal improvement. The experimental results demonstrate that the N-terminal trimethylammonium derivatives of peptides exhibit MALDI signals comparable to or exceeding those of arginine-containing standards such as angiotensin I. The advantages and limitations of the in vacuo methylation procedure are discussed.

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

An evaluation of the utility of in vacuo methylation for mass‐spectrometry‐based analyses of peptides

Laremore

Weber

Choma

2005

“…To overcome these problems, chemical modifications at the N-terminus of peptides have been attempted for more regular and 'informative' fragmentation. [14] The introduction of a positively charged group, such as quaternary or tertiary ammonium and quaternary phosphonium groups, at the N-terminus is known to enhance the relative intensity of N-terminal fragment ions (b-ions) in product ion spectra, significantly facilitating the formation of whole series of b-ions. [15][16][17][18] Similarly, it has been reported that the introduction of a negatively charged group at the N-terminus enhances the generation of C-terminal fragment ions (y-ions) with suppression of the occurrence of b-ions.…”

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confidence: 99%

Improving peptide fragmentation by N‐terminal derivatization with high proton affinity

Miyashita

Hanai

Awane

et al. 2011

An improved method of de novo peptide sequencing based on mass spectrometry using novel N-terminal derivatization reagents with high proton affinity has been developed. The introduction of a positively charged group into the N-terminal amino group of a peptide is known to enhance the relative intensity of b-ions in product ion spectra, allowing the easy interpretation of the spectra. However, the physicochemical properties of charge derivatization reagents required for efficient fragmentation remain unclear. In this study, we prepared several derivatization reagents with high proton affinity, which are thought to be appropriate for peptide fragmentation under low-energy collision-induced dissociation (CID) conditions, and examined their usefulness in de novo peptide sequencing. Comparison of the effects on fragmentation among three derivatization reagents having a guanidino or an amidino moiety, which differ in proton affinity, clearly indicated that there was an optimal proton affinity for efficient fragmentation of peptides. Among reagents tested in this study, derivatization with 4-amidinobenzoic acid brought about the most effective fragmentation. This derivatization approach will offer a novel de novo peptide sequencing method under low-energy CID conditions.

“…Many derivatization techniques have been introduced 1, 2. Several efforts focused on the simplification of peptide fragmentation patterns.…”

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confidence: 99%

N‐terminal H₃/D₃‐acetylation for improved high‐throughput peptide sequencing by matrix‐assisted laser desorption/ionization mass spectrometry with a time‐of‐flight/time‐of‐flight analyzer

Noga

Asperger

Silberring

2006

A novel method for peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) is presented. A stable isotope label introduced in the peptide N-terminus by derivatization, using a 1:1 mixture of acetic anhydride and deuterated acetic anhydride, allows for easy and unambiguous identification of ions belonging either to the N- or the C-terminal ion series in the product ion spectrum, making sequence assignment significantly simplified. The good performance of this technique was shown by successful sequencing of the contents of several peptide maps. A similar approach was recently applied to nanoelectrospray ionization (nanoESI) and nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS). The MALDI-TOF/TOF technique allows for fast, direct sequencing of modified peptides in proteomics samples, and is complementary to the nanoESI and nanoLC/MS/MS approaches.