Gas phase derivatization in peptide analysis I: the utility of trimethyl borate in identifying phosphorylation sites

Gronert, Scott; Huang, Renee; Li, Kathy H.

doi:10.1016/j.ijms.2003.10.006

Cited by 23 publications

(30 citation statements)

References 36 publications

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“…CAD of this ion leads to the loss of the third CH 3 OH molecule (Scheme 3) to give an ion with an m/z value that is eight units greater than the parent ion ( 11 B containing ions). This pattern is the same one seen in our earlier work [22], and CAD on the "ϩ8" ions leads to a breakdown of the peptide. For VsSF, CAD of the boron derivatized ion leads to the spectrum in Figure 2b.…”

Section: Vssfsupporting

confidence: 88%

“…To test the generality of the reagents, we have considered two other phosphopeptides that contain a phosphorylated and unphosphorylated serine, LsGASA and VSGAsA [22].…”

Section: Methodsmentioning

confidence: 99%

“…However, electron-capture dissociation is less prone to this problem [14,15,18]. Recently we showed that treatment with trimethoxyborane (TMB) in the gas-phase before fragmentation has a strong influence on the breakdown of phosphopeptides and can lead to more fragments that retain the phosphate [21,22]. As a result, boron derivatization provides a novel approach for gaining additional information about phosphorylation sites.…”

mentioning

confidence: 99%

“…This situation provides a reasonably demanding test of a method's ability to identify a phosphorylation site. In addition, we have included two peptides from previous work, VSGAsA and LsGASA [22]. Three boron reagents have been used: trimethoxyborane (TMB), diisopropoxymethylborane (DIPM), and diethylmethoxyborane (DEMB).…”

mentioning

confidence: 99%

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Manipulating the fragmentation patterns of phosphopeptides via gas-phase boron derivatization: Determining phosphorylation sites in peptides with multiple serines

Gronert

Horiuchi

2005

J. Am. Soc. Mass Spectrom.

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Trivalent boron species readily react with protonated phosphopeptides to give addition products with the loss of boron ligands. In the present study, trimethoxyborane (TMB), diisopropoxymethylborane (DIPM), and diethylmethoxyborane (DEMB) were allowed to react with four phosphopeptides, VsSF, LSsF, LsGASA, and VSGAsA (lower-case s indicates phosphoserine). Each of the phosphopeptides contains one serine that is phosphorylated and one that is not. Under collision-activated dissociation (CAD) conditions, the boron-derivatized peptides give fragmentation patterns that differ significantly from that of the protonated phosphopeptide. The patterns vary, depending on the number of labile (i.e., alkoxy) ligands on the boron. In general, boron derivatization increases the yield of phosphate-containing sequence ions, but dramatic effects are only seen with certain reagent/peptide combinations. However, the suite of reagents provides a means of altering and increasing the information content of phosphopeptide CAD spectra. (J Am Soc Mass Spectrom 2005, 16, 1905-1914) © 2005 American Society for Mass Spectrometry P hosphorylation is a critical post-translational modification in proteins and has been the subject of intense interest in recent years [1][2][3][4]. It is an important control mechanism of cellular activity and plays a variety of other roles in biological systems. Phosphorylation levels are often low, so highly sensitive analytical techniques, such as mass spectrometry, are needed [5]. Although mass analysis of the peptides derived from the digestion of phosphoproteins is a powerful tool for identifying phosphorylation sites [6 -18], it gives ambiguous results if the peptide contains more than one of the commonly phosphorylated amino acids, serine, threonine, or tyrosine. Identification of the phosphorylation site in these cases requires fragmentation of the peptide and the CAD process with phosphopeptides is plagued by H 3 PO 4 loss rather than sequence ion formation, which greatly complicates the analysis [19,20]. However, electron-capture dissociation is less prone to this problem [14,15,18]. Recently we showed that treatment with trimethoxyborane (TMB) in the gas-phase before fragmentation has a strong influence on the breakdown of phosphopeptides and can lead to more fragments that retain the phosphate [21,22]. As a result, boron derivatization provides a novel approach for gaining additional information about phosphorylation sites. More recently, others have used boron derivatization to analyze sugars [23,24] and nucleotide [25].In the present work, we begin with a computational study focused on the competition between H 3 PO 4 loss and peptide cleavage in a model peptide, the N-methyl amide of Gs (lower-case s indicates phosphoserine). Next, experiments are presented that illustrate how the nature of the boron reagent as well as subtle variations in the phosphopeptide structure can have dramatic effects on the fragmentation behavior during CAD. The results suggest that derivatization experiments can be tailo...

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Section: Vssfsupporting

confidence: 88%

“…To test the generality of the reagents, we have considered two other phosphopeptides that contain a phosphorylated and unphosphorylated serine, LsGASA and VSGAsA [22].…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Manipulating the fragmentation patterns of phosphopeptides via gas-phase boron derivatization: Determining phosphorylation sites in peptides with multiple serines

Gronert

Horiuchi

2005

J. Am. Soc. Mass Spectrom.

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“…Side-chain fragmentation of phosphopeptides in CID may be decreased by gas-phase derivatization using trimethyl borate prior to the fragmentation [71]. In CID three methanol molecules are lost leaving a [M 1 B 2 2H] 1 -ion.…”

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confidence: 99%

State-of-the-art in phosphoproteomics

2005

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Presently, phosphorylation of proteins is the most studied and best understood PTM. However, the analysis of phosphoproteins and phosphopeptides is still one of the most challenging tasks in contemporary proteome research. Since not every phosphoprotein is accessible by a certain method and identification of the phosphorylated amino acid residue is required in the majority of cases, various strategies for the detection and localization of phosphorylations have been developed. Identification and localization of protein phosphorylations is mostly done by MS nowadays but phosphoproteins and -peptides are often suppressed in comparison to the unphosphorylated species if measured in complex mixtures. Thus, the isolation of pure phosphopeptide samples is a main task. This review gives an overview over the most frequently used methods in isolation and detection of phosphoproteins and -peptides such as specific enrichment or separation strategies as well as the localization of the phosphorylated residues by various mass spectrometric techniques.

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Borononucleotides as Substrates/Binders for Human NMP Kinases: Enzymatic and Spectroscopic Evaluation

et al. 2012

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Borononucleotides are a family of natural nucleotide monophosphate analogues with a 5'-boronic acid function. As B-O-P linkages are known to be unstable in solution, we evaluated the ability of borononucleotides to be recognized by nucleoside monophosphate kinases and eventually foil the phosphorylation process. In this context, and with the idea of probing the influence of their size, shape, and flexibility, a library of borononucleotides were synthetized starting from the borononucleotide analogue of thymidine, which was shown to behave as a slow substrate of human TMP kinase. This study thus constitutes a good starting point for the development of new monophosphate mimics as potential substrates or ligands for NMP kinases.

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Gas phase derivatization in peptide analysis I: the utility of trimethyl borate in identifying phosphorylation sites

Cited by 23 publications

References 36 publications

Manipulating the fragmentation patterns of phosphopeptides via gas-phase boron derivatization: Determining phosphorylation sites in peptides with multiple serines

Manipulating the fragmentation patterns of phosphopeptides via gas-phase boron derivatization: Determining phosphorylation sites in peptides with multiple serines

State-of-the-art in phosphoproteomics

Borononucleotides as Substrates/Binders for Human NMP Kinases: Enzymatic and Spectroscopic Evaluation

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