pH/Organic Solvent Double-Gradient Reversed-Phase HPLC

Wiczling, Paweł; Markuszewski, Michał J.; Kaliszan, Michał; Kaliszan, Roman

doi:10.1021/ac049092r

Cited by 41 publications

(27 citation statements)

References 19 publications

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“…We firstly employed high pH (pH 10) RP-LC (13,24) to decrease the complexity of the tryptic digest of the HeLa total cell lysate. We collected 24 fractions in the first dimension RP-LC of a 72-min gradient.…”

Section: Resultsmentioning

confidence: 99%

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A Fast Workflow for Identification and Quantification of Proteomes

Chen

Jiang

Wei

et al. 2013

Molecular & Cellular Proteomics

100

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The current in-depth proteomics makes use of long chromatography gradient to get access to more peptides for protein identification, resulting in covering of as many as 8000 mammalian gene products in 3 days of mass spectrometer running time. Here we report a fast sequencing (Fast-seq) workflow of the use of dual reverse phase high performance liquid chromatography -mass spectrometry (HPLC-MS) with a short gradient to achieve the same proteome coverage in 0.5 day. We adapted this workflow to a quantitative version (Fast quantification, Fast-quan) that was compatible to large-scale protein quantification. We subjected two identical samples to the Fast-quan workflow, which allowed us to systematically evaluate different parameters that impact the sensitivity and accuracy of the workflow. Using the statistics of significant test, we unraveled the existence of substantial falsely quantified differential proteins and estimated correlation of false quantification rate and parameters that are applied in label-free quantification. We optimized the setting of parameters that may substantially minimize the rate of falsely quantified differential proteins, and further applied them on a real biological process. With improved efficiency and throughput, we expect that the Fast-seq/Fast-quan workflow, allowing pair wise comparison of two proteomes in 1 day may make MS available to the masses and impact biomedical research in a positive way. Molecular & Cellular Proteomics 12: 10.1074/mcp.M112.025023, 2370-2380, 2013.The performance of mass spectrometry has been improved tremendously over the last few years (1-3), making mass spectrometry-based proteomics a viable approach for largescale protein analysis in biological research. Scientists around the world are striving to fulfill the promise of identifying and quantifying almost all gene products expressed in a cell line or tissue. This would make mass spectrometry-based protein analysis an approach that is compatible to the second-generation mRNA deep-seq technique (4, 5).Two liquid chromatography (LC)-MS strategies have been employed to achieve deep proteome coverage. One is a single run with a long chromatography column and gradient to take advantage of the resolving power of HPLC to reduce the complexity of peptide mixtures; the other is a sequential run with two-dimensional separation (typically ion-exchange and reverse phase) to reduce peptide complexity. It was reported by two laboratories that 2761 and 4500 proteins were identified with a 10 h chromatography gradient on a dual pressure linear ion-trap orbitrap mass spectrometer (LTQ Orbitrap Velos)(6 -8). Similarly, 3734 proteins were identified using a 8 h gradient on a 2 m long column with a hybrid triple quadrupole -time of flight (Q-TOF, AB sciex 5600 Q-TOF)(9) mass spectrometer. The two-dimensional approach has yielded more identification with longer time. For example, 10,006 proteins (representing over 9000 gene products, GPs) 1 were identified in U2OS cell (10), and 10,255 proteins (representing 9207 GPs) from HeL...

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Section: Resultsmentioning

confidence: 99%

“…Previous studies have revealed the outstanding capability of 2-D RP-RP system in peptide separation (13,24,37). RP-RP approaches using two different pH values were further demonstrated to have good potential in fractionating complex protein mixtures (14,38,39).…”

Section: Discussionmentioning

confidence: 99%

A Fast Workflow for Identification and Quantification of Proteomes

Chen

Jiang

Wei

et al. 2013

Molecular & Cellular Proteomics

100

View full text Add to dashboard Cite

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“…Certain types of multi-modal separation are truly multidimensional. One example was running a simultaneous pH and organic gradient across a RP-HPLC column (Wiczling et al, 2005) so that all analytes were subjected to both mechanisms. This is one of the few techniques where the analytes are simultaneously subjected to both separations in a two-dimensional separation.…”

Section: Planarmentioning

confidence: 99%

Comprehensive multi‐dimensional liquid chromatographic separation in biomedical and pharmaceutical analysis: a review

Dixon

Pitfield

Perrett

2006

Biomedical Chromatography

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‘Multi‐dimensional’ liquid separations have a history almost as long as chromatography. In multi‐dimensional chromatography the sample is subjected to more than one separation mechanism; each mechanism is considered an independent separation dimension. The separations can be carried out either offline via fraction collection, or directly coupled online. Early multi‐dimensional separations using combinations of paper chromatography, electrophoresis and gels, in both planar and columnar modes are reviewed. Developments in HPLC have increased the number of measurable analytes in ever more complex matrices, and this has led to the concept of ‘global metabolite profiling’. This review focuses on the theory and practice of modern ‘comprehensive’ multi‐dimensional liquid chromatography when applied to biomedical and pharmaceutical analysis. Copyright © 2006 John Wiley & Sons, Ltd.

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“…Reversed-phase high-performance liquid chromatography (RP HPLC) allows determination of pK a and lipophilicity related parameters (chromatographic measure of hydrophobicity, log k w ) [5][6][7][8][9][10][11][12][13]. Lipophilicity and dissociation constant determination by HPLC have already been described in literature.…”

Section: Introductionmentioning

confidence: 99%