Automated multi-dimensional liquid chromatography: sample preparation and identification of peptides from human blood filtrate

Machtejevas, Egidijus; John, Harald; Wagner, Karl W.; Ständker, Ludger; Markó-Varga, György; Forssmann, W. G.; Bischoff, Rainer; Unger, Klaus K.

doi:10.1016/j.jchromb.2003.07.015

Cited by 60 publications

(32 citation statements)

References 31 publications

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“…Subsequent peptide separation in the second dimension was performed in parallel on four short RP columns (Micra ODS; Eichrom Technologies) implemented via column switching. Excellent reproducibility and high sample-throughput are important advantages of this fully automated technique which has high potential for quantitative LC-MS of peptides [98].…”

Section: Perspectives For On-line Proceduresmentioning

confidence: 99%

Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography?mass spectrometry

John¹,

Walden²,

Schäfer³

et al. 2004

Analytical and Bioanalytical Chemistry

158

104

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Peptide quantification by liquid chromatography-mass spectrometry (LC-MS) combines the high resolving power of reversed-phase (RP) chromatography with the excellent selectivity and sensitivity of mass spectrometric detection. On the basis of comprehensive practical experience in the analysis of small molecules, pharmaceutical research is developing technologies for analysis of a growing number of peptidic drug candidates. This article is a detailed review of procedures based on LC-MS techniques for quantitative determination of peptides. With the focus on pharmaceutical applications several technologies for sample preparation, various aspects of peptide chromatography, important characteristics of ESI-MS, selectivity of MS-detection modes, the large variability of internal standards, and modern instrumentation are discussed. The demand for reliable, robust, sensitive, and accurate methods is discussed using numerous examples from the literature, complemented by experiments and results from our laboratory.

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Section: Perspectives For On-line Proceduresmentioning

confidence: 99%

Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography?mass spectrometry

John¹,

Walden²,

Schäfer³

et al. 2004

Analytical and Bioanalytical Chemistry

158

104

View full text Add to dashboard Cite

show abstract

“…With four columns in this dimension, while one is loaded with eluate, one is regenerated and two are subjected to the gradient. Biological samples were separated (Machtejevas et al, 2004;Wagner et al, 2002). A complex MDLC system with four second-dimension columns, and online sample preparation before the first dimension (Machtejevas et al, 2004).…”

Section: Hplc-based Methodsmentioning

confidence: 99%

“…Biological samples were separated (Machtejevas et al, 2004;Wagner et al, 2002). A complex MDLC system with four second-dimension columns, and online sample preparation before the first dimension (Machtejevas et al, 2004). This figure is available in colour online at www.interscience.wiley.com/journal/bmc Anion exchange chromatography in the first dimension was coupled to parallel RP second-dimension columns with a 10-port valve for separation of proteins.…”

Section: Hplc-based Methodsmentioning

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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“…The basis of the RP mode is the hydrophobic interaction between peptides and the stationary phase. The most common stationary phase is C 18 covalently bound to a base silica material; these phases are called RP, C 18 , or octadecyl silica (better known as ODS).…”

Section: Standard Combinations In Shotgun Strategiesmentioning

confidence: 99%

“…A variety of first dimensions, not all of them LC-based, have been used, including size-exclusion chromatography (SEC), strong-cation exchange (SCX), strong-anion exchange, SDS-PAGE, and isoelectric focusing (IEF) techniques (e.g., immobilized pH gradient IEF and capillary IEF). 3,9,10,14,15,[18][19][20][21][22] Several factors are important for the first dimensionsit should have a large loading capacity, be configurable with the second dimension, and have solvent compatibility with the second mode. Several of the first-dimension methods fit these criteria, whereas others are better suited for off-line applications.…”

Section: Standard Combinations In Shotgun Strategiesmentioning

confidence: 99%

Multidimensional LC Separations in Shotgun Proteomics

2008

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Two modes of separation coupled with MS enable researchers to study complicated biological structures.Proteins are the molecular products of genes and play a central role in many biological processes. Protein expression occurs as a function of cellular and environmental conditions, and consequently proteins are expressed at different times and under different conditions. Insight is thus garnered by determining how protein expression has changed in a biological context.Over the past two decades, MS has become an important tool for the analysis of proteins. 1,2 One current method for the analysis of protein mixtures is proteolytic digestion followed by LC/MS/ MS. Once hard-to-handle proteins are converted into chemically well-behaved peptides, the approach overcomes many difficulties associated with protein mixture analysis. 3 Tandem MS has been particularly effective because the data can be directly used to identify peptides and subsequently infer which proteins (digested or undigested) are in the mixture. 4 This type of approach for the analysis of protein mixtures is often referred to as "shotgun" proteomics ( Figure 1).Because increasingly complicated biological structures are studied by tandem MS, the need for more powerful and highly resolving separation methods has grown. Consequently, the development and use of multidimensional LC (MDLC) in proteomics has thrived over the past few years. MDLC combines two or more forms of LC to increase the peak capacity, and thus the resolving power, of separations to better fractionate peptides prior to entering the mass spectrometer.High-resolution separation serves two functions in combination with MS. It better resolves peptides differing in charge and hydrophobicity to minimize ion suppression and improve ionization efficiency, and it simplifies the complexity of peptide ions entering the mass spectrometer to minimize undersampling. This last aspect is important because the tandem MS process is driven by data-dependent data acquisition and has a finite cycle time. A higher peak capacity and better resolving power improve the acquisition of data and can lead to a better representation of the proteins in the mixture. Improved resolution also results in a larger dynamic range.Although MDLC is by no means a new concept and has a long history, it has been enjoying a renaissance in proteomics. 5 In this article, we will provide an overview of the background, important applications, and future prospects for MDLC; in particular, we will focus on applications involving peptides. (To listen to a podcast about this feature, please go to the Analytical Chemistry website at pubs.acs.org/ac.)

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Automated multi-dimensional liquid chromatography: sample preparation and identification of peptides from human blood filtrate

Cited by 60 publications

References 31 publications

Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography?mass spectrometry

Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography?mass spectrometry

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

Multidimensional LC Separations in Shotgun Proteomics

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