Intact protein analysis by matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry

Lin, Melanie; Campbell, J. M.; Mueller, Dieter; Wirth, Urs

doi:10.1002/rcm.1102

Cited by 65 publications

(77 citation statements)

References 13 publications

(17 reference statements)

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“…In all experiments, 500 fmol of the derivatized peptide was applied on the MALDI stainless steel probe. The minimum amount of protein required to obtain good quality MS/MS spectra varies from 10 pmol to 200 fmol, as recently reported by Lin et al [24]. Fragmentation spectra were obtained from approximately 5000 laser shots of 500 fmol of the peptide.…”

Section: Tof/tof Analysis Of Native and Derivatized Peptidesmentioning

confidence: 98%

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

Samyn

Debyser

Sergeant

et al. 2004

J. Am. Soc. Mass Spectrom.

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The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have increased its application in recent years. The most common method of "peptide mass fingerprint" analysis often does not provide robust identification. Additional sequence information, obtained by post-source decay or collision induced dissociation, provides additional constraints for database searches. However, de novo sequencing by mass spectrometry is not yet common practice, most likely because of the difficulties associated with the interpretation of high and low energy CID spectra. Success with this type of sequencing requires full sequence coverage and demands better quality spectra than those typically used for data base searching. In this report we show that full-length de novo sequencing is possible using MALDI TOF/TOF analysis. The interpretation of MS/MS data is facilitated by N-terminal sulfonation after protection of lysine side chains (Keough et al., Proc. Natl. Acad. Sci. U.S. A. 1999, 96, 7131-7136). Reliable de novo sequence analysis has been obtained using sub-picomol quantities of peptides and peptide sequences of up to 16 amino acid residues in length have been determined. The simple, predictable fragmentation pattern allows routine de novo interpretation, either manually or using software. Characterization of the complete primary structure of a peptide is often hindered because of differences in fragmentation efficiencies and in specific fragmentation patterns for different peptides. These differences are controlled by various structural parameters including the nature of the residues present. The influence of the presence of internal Pro, acidic and basic residues on the TOF/TOF fragmentation pattern will be discussed, both for underivatized and guanidinated/sulfonated peptides. (J Am Soc Mass Spectrom 2004, 15, 1838 -1852) © 2004 American Society for Mass Spectrometry P rotein identification protocols in proteomics currently rely on the peptide mass fingerprinting (PMF) technique in which a (mostly gel-purified) protein is digested with an endoprotease of known cleavage specificity. The masses of the resulting peptides are measured by mass spectrometry, usually matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), and matched to peptide masses that have been generated theoretically from proteins in databases. Notwithstanding the fact that the PMF approach is useful for identifying proteins in simple mixtures (e.g., 2D-PAGE gel spots), the identification of proteins in more complex mixtures often requires partial peptide sequence data obtained by tandem mass spectrometry (MS/MS) methods. If accurate genome sequence information is available, database search algorithms can be used in conjunction with MS/MS to readily identify proteins. For proteins not contained within sequence databases, it is necessary to determine partial or complete amino acid sequences using either manual or automated de novo peptide sequence analysis methods. This genera...

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Section: Tof/tof Analysis Of Native and Derivatized Peptidesmentioning

confidence: 98%

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

Samyn

Debyser

Sergeant

et al. 2004

J. Am. Soc. Mass Spectrom.

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“…The amino acid sequence of the B-subunit of Stx2a is shown above the spectrum. PSD of low charge state protein ions (e.g., +1 or +2) are known to fragment on the C-terminal side of aspartic acid (D) and glutamic acid (E) residues and on the N-terminal side of proline (P) residues [36,37]. The favorability of fragmentation at D-and E-residues is the result of transfer of their acidic protons from their side chains to the polypeptide backbone, resulting in a weakening of the amide bond [36,37].…”

Section: Resultsmentioning

confidence: 99%

“…PSD of low charge state protein ions (e.g., +1 or +2) are known to fragment on the C-terminal side of aspartic acid (D) and glutamic acid (E) residues and on the N-terminal side of proline (P) residues [36,37]. The favorability of fragmentation at D-and E-residues is the result of transfer of their acidic protons from their side chains to the polypeptide backbone, resulting in a weakening of the amide bond [36,37]. As expected, the most abundant fragment ions in Figure 4a (y 46 , y 53 , y 54 ) are the result of polypeptide backbone fragmentation on the C-terminal side of D residues (except for those proximal to the N-or C-termini).…”

Section: Resultsmentioning

confidence: 99%

“…In addition, the two amino acid substitutions that distinguish the B-subunit sequence of Stx2a from Stx2c both involve an aspartic acid (D) residue, which not only result in differences in fragment ion m/z but also significantly alters the fragmentation channels of these protein ions. Fragmentation of the polypeptide backbone is highly favored on the C-terminal side of D-residues [36,37] and this facile fragmentation channel is clearly demonstrated in Figure 4 and Supplementary Figure 1.…”

Section: Top-down Versus Bottom-up Proteomic Analysis For Distinguishmentioning

confidence: 93%

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Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

Fagerquist

Zaragoza

2015

J. Am. Soc. Mass Spectrom.

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Abstract. We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H-strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) with post source decay (PSD) and real time-quantitative polymerase chain reaction (RT-qPCR). Stx2a and Stx2c in STEC strain E32511 were quantified from the integrated peak area of their singly charged disulfide-intact B-subunit ions at m/z 7819 and m/z~7774, respectively. We found that the Stx2a subtype was 21-fold more abundant than the Stx2c subtype. The two amino acid substitutions (16D ↔ 16 N and 24D ↔ 24A) that distinguish Stx2a from Stx2c not only result in a mass difference of 45 Da between their respective B-subunits but also result in distinctly different fragmentation channels by MS/MS-PSD because both substitutions involve an aspartic acid (D) residue. Importantly, these two substitutions have also been linked to differences in subtype toxicity. We measured the relative abundances of mRNA transcripts using RT-qPCR and determined that the stx2a transcript is 13-fold more abundant than stx2c transcript. In silico secondary structure analysis of the full mRNA operons of stx2a and stx2c suggest that transcript structural differences may also contribute to a relative increase of Stx2a over Stx2c. In consequence, toxin expression may be under both transcriptional and post-transcriptional control.

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“…11,12 The advantage of using matrix-assisted laser desorption/ionization (MALDI)-MS/MS for IgG2 disulfide-bonded (DSB) peptide analysis is the predominance of singly-charged ions in the MALDI spectra, which considerably simplifies data analysis and renders it compatible with both semi-quantitative output and automation from a single LC-MS/MS analysis. 36–38 …”

Section: Introductionmentioning

confidence: 99%

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

Resemann

Liu-Shin

Tremintin

et al. 2018

mAbs

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Human antibodies of the IgG2 subclass exhibit complex inter-chain disulfide bonding patterns that result in three structures, namely A, A/B, and B. In therapeutic applications, the distribution of disulfide isoforms is a critical product quality attribute because each configuration affects higher order structure, stability, isoelectric point, and antigen binding. The current standard for quantification of IgG2 disulfide isoform distribution is based on chromatographic or electrophoretic techniques that require additional characterization using mass spectrometry (MS)-based methods to confirm disulfide linkages. Detailed characterization of the IgG2 disulfide linkages often involve MS/MS approaches that include electrospray ionization or electron-transfer dissociation, and method optimization is often cumbersome due to the large size and heterogeneity of the disulfide-bonded peptides. As reported here, we developed a rapid LC-MALDI-TOF/TOF workflow that can both identify the IgG2 disulfide linkages and provide a semi-quantitative assessment of the distribution of the disulfide isoforms. We established signature disulfide-bonded IgG2 hinge peptides that correspond to the A, A/B, and B disulfide isoforms and can be applied to the fast classification of IgG2 isoforms in heterogeneous mixtures.

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Intact protein analysis by matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry

Cited by 65 publications

References 13 publications

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

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