Development of methods for the charge-derivatization of peptides in polyacrylamide gels and membranes for their direct analysis using matrix-assisted laser desorption-ionization mass spectrometry

Strahler, John R.; Smelyanskiy, Yanina; Lavine, Gary; Allison, John

doi:10.1016/s0168-1176(97)00221-8

Cited by 17 publications

(11 citation statements)

References 22 publications

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“…The TMPP-Ac reagent has been used to derivatize protein digest mixtures at the 25-pmol level (Huang et al, 1997b). This reagent was also used to derivatize peptides located in an electrophoresis gel or a transfer membrane (Strahler et al, 1997). The modified peptides were analyzed by MALDI and MALDI-PSD directly from the gel or membrane on a modified sample plate.…”

Section: Charge-derivatized Peptides ▪mentioning

confidence: 99%

Charge derivatization of peptides for analysis by mass spectrometry

Roth¹,

Huang²,

Sadagopan³

et al. 1998

Mass Spectrom. Rev.

133

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Section: Charge-derivatized Peptides ▪mentioning

confidence: 99%

Charge derivatization of peptides for analysis by mass spectrometry

Roth¹,

Huang²,

Sadagopan³

et al. 1998

Mass Spectrom. Rev.

133

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“…A protonated Arg or Lys or a fixed-charge group near the C terminus enhances fragment ions containing the C terminus of the peptide (x, y, z ions). It is not surprising, therefore, that several researchers have used N-terminal fixed-charge derivatives (quaternary ammonium and phosphonium groups) to improve MALDI sensitivity and to try to direct and simplify peptide fragmentation patterns observed in PSD mass spectra (28)(29)(30)(31)(32)(33)(34)(35). In our hands, generally poor-quality MALDI PSD mass spectra have been obtained with fixed positive-charge derivatives.…”

mentioning

confidence: 99%

A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry

Keough

Youngquist

Lacey

1999

Proc. Natl. Acad. Sci. U.S.A.

225

262

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A method has been developed for de novo peptide sequencing using matrix-assisted laser desorption ionization mass spectrometry. This method will facilitate biological studies that require rapid determination of peptide or protein sequences, e.g., determination of posttranslational modifications, identification of active compounds isolated from combinatorial peptide libraries, and the selective identification of proteins as part of proteome studies. The method involves fast, one-step addition of a sulfonic acid group to the N terminus of tryptic peptides followed by acquisition of postsource decay (PSD) fragment ion spectra. The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions). The overall method has been applied to pmol quantities of peptides. The resulting PSD fragment ion spectra often exhibit uninterrupted sequences of 20 or more amino acid residues. However, fragmentation efficiency decreases considerably at amide bonds on the C-terminal side of Pro. The spectra are simple enough that de novo sequence tagging is routine. The technique has been successfully applied to peptide mixtures, to high-mass peptides (up to 3,600 Da) and to the unambiguous identification of proteins isolated from two-dimensional gel electrophoresis. The PSD spectra of these derivatized peptides often allow far more selective protein sequence database searches than those obtained from the spectra of native peptides.Knowledge of protein sequences is fundamentally important for understanding many physiological processes at the molecular level (1). Tandem mass spectrometry has become an increasingly essential tool for protein and peptide sequencing because of its speed, sensitivity, and applicability to complex mixtures (2). Recently, postsource decay (PSD) matrix-assisted laser desorption ionization (MALDI) mass spectrometry was developed for high-sensitivity peptide sequencing applications (3-8). Subpicomole limits of analysis were reported as a result of the high yield of fragment ions and the high ion transmission inherent with time-of-flight mass spectrometry (5). Kaufmann et al. (5) also noted several problems associated with PSD MALDI sequencing of peptides, including the complexity of the resulting fragmentation patterns and the lack of computer algorithms capable of interpreting the complex spectra. The recent incorporation of delayed extraction (DE) (9, 10), a technique designed to improve precursor-ion mass resolution and mass-measurement accuracy, reduced the rate of PSD fragmentation by at least an order of magnitude. As a result, many low-intensity precursor ions obtained by DE MALDI do not produce enough PSD fragmentation to allow derivation of even short sequence tags (7).Positively charged derivatives have been used in desorption mass spectrometry for many years to improve sensitivity by enhancing ionization efficiencies (11, 12...

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“…Derivatization with labels containing a dansyl11 or coumarin core12, 13 attached to the N‐terminus was shown to improve the sensitivity for peptides having low ionization yields. Activated esters containing a fixed charge, S ‐pentafluorophenyl[tris(2,4,6‐triethoxyphenyl)phosphonium]acetate bromide (TMPP‐AcSC 6 F 5 bromide), were used for N‐terminal derivatization in order to increase signal intensities in MALDI‐MS analysis 14, 15. Similarly, signal enhancement was observed by charge‐localized derivatization with triphenylphosphonium 16.…”

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confidence: 99%

Derivatization by 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate for enhancing the ionization yield of small peptides and glycopeptides in matrix‐assisted laser desorption/ionization and electrospray ionization mass spectrometry

Ullmer

Plematl

Rizzi

2006

Rapid Comm Mass Spectrometry

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The characterization of glycosylation in proteins by mass spectrometry (MS) is often impeded by strong suppression of ionization of glycopeptides in the presence of non-glycosylated peptides. Glycopeptides with a large carbohydrate part and a short peptide backbone are particularly affected by this problem. To meet the goal of generating mass spectra exhibiting glycopeptide coverages as complete as possible, derivatization of glycopeptides offers a practical way to increase their ionization yield. This paper investigated derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) which is a rapid labeling technique commonly used for fluorescence detection in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). As test samples we used peptides and glycopeptides obtained by enzymatic digestion of three different glycoproteins, i.e., human antithrombin, chicken ovalbumin, and bovine alpha1-acid-glycoprotein. It was found that AQC derivatization resulted in strongly increased signal intensities when analyzing small peptides and glycopeptides by matrix-assisted laser desorption/ionization (MALDI)-MS. For these compounds the limit of detection could be reduced to low fmol amounts. Without derivatization only glycopeptides containing large peptide backbones were detected by MALDI-MS. This effect was even significant when glycopeptides were pre-separated and enriched by means of lectin affinity chromatography before MALDI-MS analysis and when using electrospray ionization (ESI). This labeling method, applied in combination with MS detection for the first time, was found to be well suited for the enhancement of detection sensitivity for small glycopeptides in MALDI-MS analysis and thus for reducing the need for pre-separation steps.

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Development of methods for the charge-derivatization of peptides in polyacrylamide gels and membranes for their direct analysis using matrix-assisted laser desorption-ionization mass spectrometry

Cited by 17 publications

References 22 publications

Charge derivatization of peptides for analysis by mass spectrometry

Charge derivatization of peptides for analysis by mass spectrometry

A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry

Derivatization by 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate for enhancing the ionization yield of small peptides and glycopeptides in matrix‐assisted laser desorption/ionization and electrospray ionization mass spectrometry

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