A simple and highly successful C‐terminal sequence analysis of proteins by mass spectrometry

Kuyama, Hiroki; Shima, Keisuke; Sonomura, Kazuhiro; Yamaguchi, Minoru; Ando, Eiji; Nishimura, Osamu; Tsunasawa, Susumu

doi:10.1002/pmic.200701044

Cited by 41 publications

(40 citation statements)

References 21 publications

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“…TMPP-mass tag serves as a reporter ion signal (m/z 589) in MS/MS measurement. K* indicates formylated lysine application of the present method to pigeon cytochrome c, to which conventional approaches involving the digestion with trypsin [12,13] or LysC [6] were very difficult to apply due to the presence of lysine at the C termini. That is, the digestion of this protein with trypsin or LysC produces a mixture of peptides, each of which has arginine (only with trypsin) or lysine at the C terminus.…”

Section: C-terminal Sequencing Of Proteins and Choice Of Proteolytic mentioning

confidence: 99%

“…To address the issue of C-terminal specific analysis, we suggested a stratagem [6] consisting of a combination of LysC digestion in conjunction with α-amino-specific derivatization with tris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) group [7,8] and with p-phenylenediisothiocyanate (DITC)-modified aminopropyl-glass beads [9], which removes peptides by capturing the remaining amino groups. A likely limitation of this approach is that its performance depends strongly on the sequence position of the lysine residue; the possibility of obtaining C-terminal peptides eligible for sequencing is not sufficiently high to envisage the wider applicability.…”

Section: Introductionmentioning

confidence: 99%

“…The C-terminal peptide sequence of cytochrome c (pigeon) was first analyzed, which was chosen on the ground that it represents one of the theoretically inapplicable proteins for the preceding sequencing strategies [6,12,13], due to the presence of lysine residue at the C terminus. Then the method was verified using four other proteins (superoxide dismutase (Escherichia coli), triosephosphate isomerase (rabbit), glyceraldehyde-3-phosphate dehydrogenase (rabbit), and hemoglobin (bovine)).…”

Section: Introductionmentioning

confidence: 99%

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C-Terminal sequencing of protein by MALDI mass spectrometry through the specific derivatization of the α-carboxyl group with 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide

et al. 2012

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We present here an approach to C-terminal sequencing of proteins by the procedure consisting of the following: (1) derivatization of the C-terminal α-carboxyl group with 3-aminopropyltris(2,4,6-trimethoxyphenyl)-phosphonium bromide (TMPP-propylamine) through oxazolone chemistry, (2) enzymatic proteolysis of the TMPP-derivatized protein, and (3) MALDI-MS/MS analysis of the peptide mixture, in which the C-terminal peptide incorporating the TMPP group is preferably detected. In this protocol, it is possible to choose any endoproteinase such as trypsin, GluC, and AspN for digestion so that a C-terminal peptide with length appropriate for mass spectrometric sequencing could be generated. The peptide labeled with TMPP-propylamine at the C terminus tends to exhibit y-type ions in MS/MS spectra, allowing manual sequence interpretation with the simplified fragmentation pattern. The efficacy of the method was verified with five proteins, which demonstrated that the C-terminal peptides were readily distinguishable by their peak intensity and characteristic mass signature peak in MALDI-PSD analysis.

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Section: C-terminal Sequencing Of Proteins and Choice Of Proteolytic mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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C-Terminal sequencing of protein by MALDI mass spectrometry through the specific derivatization of the α-carboxyl group with 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide

et al. 2012

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“…TMPP-Ac-OSu has been introduced as a reagent for N- and C-terminal sequence analysis of proteins [12, 13]. De novo sequencing of protein termini with the TMPP labeling method provides higher fidelity [14].…”

Section: Introductionmentioning

confidence: 99%

Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G

Suh

Clark

Parmer

et al. 2010

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The Staphylococcus aureus surface protein G (SasG) is an important mediator of biofilm formation in virulent S. aureus strains. A detailed analysis of its primary sequence has not been reported to date. SasG is highly abundant in the cell wall of the vancomycin-intermediate S. aureus strain HIP5827, and was purified and subjected to sequence analysis by MS. Data from MALDI-TOF and LC-MS/MS experiments confirmed the predicted N-terminal signal peptide cleavage site at residue A 51 and the C-terminal cell wall anchor site at residue T 1086 . The protein was also derivatized with N-succinimidyloxycarbonyl-methyl-tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPPAc-OSu) to assess the presence of additional N-terminal sites of mature SasG. TMPP-derivatized SasG peptides featured m/z peaks with a 572 Da mass increase over the equivalent underivatized peptides. Multiple N-terminal peptides, all of which were observed in the 150 amino acid segment following the signal peptide cleavage at the residue A 51 , were characterized from MS and MS/MS data, suggesting a series of successive N-terminal truncations of SasG. A strategy combining TMPP derivatization, multiple enzyme digestions to generate overlapping peptides and detailed MS analysis will be useful to determine and understand functional implications of PTMs in bacterial cell wallanchored proteins, which are frequently involved in the modulation of virulence-associated bacterial surface properties.

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“…We suggest here a chemical method for detecting PTMs at the C‐termini of peptides and proteins, utilizing a technique for the isolation of the C‐terminal peptides 9 in combination with a reaction for selectively derivatizing the C‐terminal carboxyl group according to oxazolone chemistry 10, 11. The scheme for C‐terminal derivatization is specifically designed to convert the free C‐terminal carboxyl group into methylamide (CONHCH 3 ), increasing the mass of the peptide by 13 Da, while peptides possessing the amide (CONH 2 ) group remain unchanged (Fig.…”

Section: Introductionmentioning

confidence: 99%

A new approach for detecting C‐terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization

et al. 2009

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We describe a mass spectrometric method for distinguishing between free and modified forms of the C-terminal carboxyl group of peptides and proteins, in combination with chemical approaches for the isolation of C-terminal peptides and site-specific derivatization of the C-terminal carboxyl group. This method could most advantageously be exploited to discriminate between peptides having C-terminal carboxyl groups in the free (COOH) and amide (CONH(2)) forms by increasing their mass difference from 1 to 14 Da by selectively converting the free carboxyl group into methylamide (CONHCH(3)). This method has been proven to be applicable to peptides containing aspartic and glutamic acids, because all the carboxyl groups except the C-terminal one are inert to derivatization, according to oxazolone chemistry. The efficiency of the method is illustrated by a comparison of the peaks of processed peptides obtained from a mixture of adrenomedullin, calcitonin, and BSA. Among these components of the mixture, only the C-terminal peptide of BSA exhibited the mass shift of 13 Da upon treatment, eventually unambiguously validating the C-terminal amide structures of adrenomedullin and calcitonin. The possibility of extending this method for the analysis of C-terminal PTMs is also discussed.

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A simple and highly successful C‐terminal sequence analysis of proteins by mass spectrometry

Cited by 41 publications

References 21 publications

C-Terminal sequencing of protein by MALDI mass spectrometry through the specific derivatization of the α-carboxyl group with 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide

C-Terminal sequencing of protein by MALDI mass spectrometry through the specific derivatization of the α-carboxyl group with 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide

Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G

A new approach for detecting C‐terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization

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