Multidimensional separation of peptides for effective proteomic analysis

Issaq, Haleem J.; Chan, King C.; Janini, George M.; Conrads, Thomas P.; Veenstra, Timothy D.

doi:10.1016/j.jchromb.2004.07.042

Cited by 189 publications

(127 citation statements)

References 67 publications

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“…The column's contribution to peak width at half height (W 1/2 ) was calculated using the following equation given by Snyder: 21 (15) where N is the isocratic plate number and G is the gradient band compression factor; k' f is the solute retention factor when the solute is at the column exit and is given by: (16) Changes in column efficiency (N) as a function of flow rate were accounted by using the van Deemter equation. The coefficients of the van Deemter equation (A, B, C) were determined from an experimental flow curve obtained for the column used in this study.…”

Section: Prediction Of Gradient Peak Widthmentioning

confidence: 99%

Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography: Fixed Column Format

et al. 2006

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The optimization of peak capacity in gradient elution RPLC is essential for the separation of multicomponent samples such as those encountered in proteomic research. In this work we study the effect of gradient time (t G ), flow rate (F), temperature (T) and final eluent strength (ϕ final ) on the peak capacity of separations of peptides that are representative of the range in peptides found in a tryptic digest. We find that there are very strong interactions between the individual variables (e.g. flow rate and gradient time) which make the optimization quite complicated. On a given column, one should first set the gradient time to the longest tolerable and then set the temperature to the highest achievable with the instrument. Next, the flow rate should be optimized using a reasonable but arbitrary value of ϕ final . Last, the final eluent strength should be adjusted so that the last solute elutes as close as possible to the gradient time. We also develop an easily implemented, highly efficient and effective Monte Carlo search strategy to simultaneously optimize all the variables. We find that gradient steepness is an important parameter that influences peak capacity and an optimum range of gradient steepness exists in which the peak capacity is maximized.

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Section: Prediction Of Gradient Peak Widthmentioning

confidence: 99%

Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography: Fixed Column Format

et al. 2006

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“…Most strategies are orthogonal to the "standard" combination of LC-MS, 4 either at the protein [5][6][7] or the peptide level. 8 For instance, peptide fractionation by SCX-LC followed by reversed-phase-LC, termed MudPit, 9 is a well-established approach for peptide separation enabling the identification of multiple thousands of peptides in a single experiment. An alternative and common way to separate proteins is SDS polyacrylamide gel electrophoresis (PAGE).…”

Section: Introductionmentioning

confidence: 99%

In-Gel Isoelectric Focusing of Peptides as a Tool for Improved Protein Identification

et al. 2006

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In the analysis of proteins in complex samples, pre-fractionation is imperative to obtain the necessary depth in the number of reliable protein identifications by mass spectrometry. Here we explore isoelectric focusing of peptides (peptide IEF) as an effective fractionation step that at the same time provides the added possibility to eliminate spurious peptide identifications by filtering for pI. Peptide IEF in IPG strips is fast and sharply confines peptides to their pI. We have evaluated systematically the contribution of pI filtering and accurate mass measurements on the total number of protein identifications in a complex protein mixture (Drosophila nuclear extract). At the same time, by varying Mascot identification cutoff scores, we have monitored the false positive rate among these identifications by searching reverse protein databases. From mass spectrometric analyses at low mass accuracy using an LTQ ion trap, false positive rates can be minimized by filtering of peptides not focusing at their expected pI. Analyses using an LTQ-FT mass spectrometer delivers low false positive rates by itself due to the high mass accuracy. In a direct comparison of peptide IEF with SDS-PAGE as a pre-fractionation step, IEF delivered 25% and 43% more proteins when identified using FT-MS and LTQ-MS, respectively. Cumulatively, 2190 non redundant proteins were identified in the Drosophila nuclear extract at a false positive rate of 0.5%. Of these, 1751 proteins (80%) were identified after peptide IEF and FT-MS alone. Overall, we show that peptide IEF allows to increase the confidence level of protein identifications, and is more sensitive than SDS-PAGE.

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“…These methods include orthogonal chromatography, where a sample is separated independently using two different stationary phases [1,2], and comprehensive two-dimensional liquid chromatography (2D-LC), where sequential fractions of the eluent from a first column are introduced into a second column [3][4][5][6][7]. Although these methods provide increased peak capacity relative to conventional LC methods, there still is a high risk of peak overlap for complex samples, such as those encountered in proteomic and metabolomic investigations [8][9][10]. Multivariate curve resolution methods such as parallel factor analysis (PARAFAC) can be used to resolve overlapped peaks and to provide quantitative information for the components of the mixture [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

“…Comprehensive two-dimensional separation techniques, including two-dimensional gas chromatography (2D-GC) and 2D-LC are becoming popular for the separation of complex mixtures [7,9,19]. Giddings defined comprehensive two-dimensional separations as those in which a sample is subjected to two separation techniques that are at right angles (or orthogonal) to one another.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the multivariate selectivity of multi‐way liquid chromatography methods

Cantwell

Porter

Rutan

2007

Journal of Chemometrics

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Multivariate figures of merit are used to characterize the sensitivity, selectivity, and precision of multi-dimensional liquid chromatography (LC) methods. Specifically in this work, we used a multivariate selectivity parameter to characterize the performance of orthogonal LC and twodimensional liquid chromatography (2D-LC) methods with and without diode array detection (DAD), and to aid in the optimization of these new separation approaches. We found that the average selectivity of 47 drug compounds was increased by as much as 58% upon the addition of a second, orthogonal separation. We also found that the selectivity for some individual drug compounds improved by more than a factor of two when DAD detection was used compared to single wavelength detection. In the comprehensive 2D-LC simulations, using a multi-channel detector increased the average selectivity of 500 components by 7.3% for a 21 s cycle time. The multivariate selectivity parameter was shown to be useful for determining the information gained when additional dimensions of data are collected on multi-dimensional instrumentation.

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Multidimensional separation of peptides for effective proteomic analysis

Cited by 189 publications

References 67 publications

Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography: Fixed Column Format

Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography: Fixed Column Format

In-Gel Isoelectric Focusing of Peptides as a Tool for Improved Protein Identification

Evaluation of the multivariate selectivity of multi‐way liquid chromatography methods

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