Quantification of Polypeptides by Mass Spectrometry

Cutillas, Pedro R.

doi:10.1002/9780470196502.ch13

Cited by 2 publications

(8 citation statements)

References 107 publications

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“…However, ion suppression from complex mixtures is reduced by using nanoliter volume/minute flow rates and increased separation gradient lengths. 43–45 Therefore in the current study, peptides were analyzed using a 300 nL/min flow rate and separated over 100 min of the gradient. In addition, as seen in Figure 2, approximately 75% of the identified peptides were found in both the ZT 6 and ZT 18 samples, suggesting that the composition of the samples generated from ZT 6 and ZT 18 are similar.…”

Section: Discussionmentioning

confidence: 99%

“…The daytime and nighttime samples contained distinct peptides, and so factors arising from these differences such as distinct coeluting analytes may have affected the quantitative accuracy (for example, from differences in ion suppression). However, ion suppression from complex mixtures is reduced by using nanoliter volume/minute flow rates and increased separation gradient lengths. − Therefore in the current study, peptides were analyzed using a 300 nL/min flow rate and separated over 100 min of the gradient. In addition, as seen in Figure , approximately 75% of the identified peptides were found in both the ZT 6 and ZT 18 samples, suggesting that the composition of the samples generated from ZT 6 and ZT 18 are similar.…”

Section: Discussionmentioning

confidence: 99%

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Quantitative Peptidomics for Discovery of Circadian-Related Peptides from the Rat Suprachiasmatic Nucleus

et al. 2013

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In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light input via the optic chiasm and synchronizes many daily biological rhythms. Here we explore variations in the expression levels of neuropeptides present in the SCN of rats using a label-free quantification approach that is based on integrating peak intensities between daytime, Zeitgeber time (ZT) 6, and nighttime, ZT 18. From nine analyses comparing the levels between these two time points, ten endogenous peptides derived from eight prohormones exhibited significant differences in their expression levels (FDR adjusted p-value <0.05). Of these, seven peptides derived from six prohormones, including GRP, PACAP, and CART, exhibited ≥30% increases at ZT 18, and the VGRPEWWMDYQ peptide derived from proenkephalin A showed a >50% increase at nighttime. Several endogenous peptides showing statistically significant changes in this study have not been previously reported to alter their levels as a function of time of day, nor have they been implicated in prior functional SCN studies. This information on peptide expression changes serves as a resource for discovering unknown peptide regulators that affect circadian rhythms in the SCN.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Quantitative Peptidomics for Discovery of Circadian-Related Peptides from the Rat Suprachiasmatic Nucleus

et al. 2013

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“…Peptides were purified and separated on a 1D-LC coupled to a QTOF instrument and quantified with DecyderMS software . Previously gathered MS/MS data − was clustered by use of the Bonanza algorithm, which allowed us to look up experimentally obtained MSI peptides in the clustering overview in order to infer their identity . FTMS measured peaks on adjacent MSI prepared tissue slices were used to align spectra of these different MS sources, matching identical peaks in the process.…”

Section: Methodsmentioning

confidence: 99%

“…These spectra were analyzed in DataAnalysis 4.0 (Bruker Daltonik GmbH, Bremen, Germany) and provided a list of highly accurately determined masses. These values allowed to recalibrate the MSI spectra and accurately match the MSI peaks with the LC-QTOF peaks on one hand and the parent ions from the clustered fragmentation data from previous analyses − on the other hand (Figure ).…”

Section: Methodsmentioning

confidence: 99%

“…For the relative quantification we specifically assessed the molecular content of the islets of Langerhans in the 1–7 kDa mass range of ob / ob and wt mice with MSI and LC/MS. We also addressed the benefits of coupling Bonanza clustering of previously gathered MS/MS spectra − to MSI data sets. Clustering MS/MS spectra retrieves information on the posttranslational modifications (PTM) of the peptidome, drastically increasing the number of identified MS/MS spectra.…”

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confidence: 99%

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Linking Mass Spectrometric Imaging and Traditional Peptidomics: A Validation in the Obese Mouse Model

Minerva

Boonen²,

Menschaert

et al. 2011

Anal. Chem.

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MALDI mass spectrometry imaging (MSI) is a promising technique in the field of molecular (immuno)histology but is confronted with the problematic large-scale identification of peptides from thin tissue sections. In this study we present a workflow that significantly increased the number of identified peptides in a given MALDI-MSI data set and we evaluated its power concerning relative peptide quantifications. Fourier transform mass spectrometry (FTMS) profiling on matrix-coated thin tissue sections allowed us to align spectra of different MS sources, matching identical peaks in the process, thus linking MSI data to tandem mass spectrometry (MS/MS) on one hand and semiquantitative liquid chromatography (LC)/MS data on the other. Bonanza clustering was applied in order to group MS/MS spectra of structurally related peptides, making it possible to infer the identity of MSI-detected compounds based on identified members within the same cluster, effectively increasing the number of identifications in a single MSI data set. Out of 136 detected peptides with MALDI-MSI, we were able to identify 46 peptides. For 31 of these, a LC/quadrupole time-of-flight (QTOF) counterpart was detected, and we observed similar obese (ob/ob) to wild-type (wt) peak intensity ratios for 18 peptides. This workflow significantly increased the number of identifications of peptide masses detected with MALDI-MSI and evaluated the power of this imaging method for relative quantification of peptide levels between experimental conditions.

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Quantification of Polypeptides by Mass Spectrometry

Cited by 2 publications

References 107 publications

Quantitative Peptidomics for Discovery of Circadian-Related Peptides from the Rat Suprachiasmatic Nucleus

Quantitative Peptidomics for Discovery of Circadian-Related Peptides from the Rat Suprachiasmatic Nucleus

Linking Mass Spectrometric Imaging and Traditional Peptidomics: A Validation in the Obese Mouse Model

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