Recent advances in peptide separation by multidimensional liquid chromatography for proteome analysis

Palma, Serena Di; Hennrich, Marco L.; Heck, Albert J. R.; Mohammed, Shabaz

doi:10.1016/j.jprot.2012.04.033

Cited by 147 publications

(145 citation statements)

References 170 publications

(304 reference statements)

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“…The quantification of the plant Golgi proteome has been considered challenging, because this organelle is proportionally of low abundance in the cell; therefore, its constituent proteins are rarely identified in conventional proteomics experiments. Investigation of such low-abundance proteins generally requires sample fractionation on the organelle, protein, or peptide level (Stasyk and Huber, 2004;Haynes and Roberts, 2007;Di Palma et al, 2012).…”

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confidence: 99%

Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

et al. 2014

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The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (dataindependent acquisition method using ion mobility separation known as LC-IMS-MS E [or HDMS E ]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle.

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confidence: 99%

Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

et al. 2014

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“…Gel-free approaches, on the other hand, employ an initial proteolytic digestion of the complex mixture with fractionation at the peptide level using multidimensional liquid chromatography. Among several multidimensional liquid chromatographic methods, 13 the most extensively used technique separates the complex peptides mixture using strong cation-exchange (SCX) chromatography, and then reversed-phase (RP) LC-MS/MS. The principle behind this is that peptides are first separated on the basis of their charge, and then on the basis of their hydrophobicity.…”

Section: Discussion Of Methodsmentioning

confidence: 99%

“…Isotope-labeled (heavy) peptides and their native (light) peptides can be easily distinguished in a mass spectrometer by their differences in mass, thus enabling accurate peptide, hence protein, quantification. Most commonly used stable isotopes are 13 C, 15 N, 2 H, and 18 O and these can be metabolically, enzymatically, or chemically incorporated into proteins or peptides. In labelincorporated methods, samples are combined after the labeling and analyzed by nanoLC-MS/MS, which essentially avoids differences induced by variations in instrument performance between measurements.…”

Section: Ms-based Quantitative Proteomics Approachesmentioning

confidence: 99%

Bottom-Up Mass Spectrometry–Based Proteomics as an Investigative Analytical Tool for Discovery and Quantification of Proteins in Biological Samples

Amunugama

Jones

Ford

et al. 2013

Advances in Wound Care

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Objective: The objective of this overview is to introduce bottom-up mass spectrometry (MS)-based proteomics approaches and strategies, widely used in other biomedical research fields, to the wound-healing research community. Approaches: Two major proteomics workflows are discussed: gel-based and gel-free chromatographic separation to reduce the complexity of the sample at protein and peptide level, respectively, prior to nano-liquid chromatographytandem mass spectrometry analysis. Other strategies to discover less abundant proteins present in the sample, are also briefly discussed along with label-free and label-incorporated methods for protein quantification. Overall, the experimental workflows are designed and continually improved to increase the number of proteins identifiable and quantifiable. Discussion: Recent advances and improvements in all areas of proteomics workflow from sample preparation, to acquisition of massive amounts of data, to bioinformatics analysis have made this technology an indispensable tool for in-depth large-scale characterization of complex proteomes. This technology has been successfully applied in studies focusing on biomarker discovery, differential protein expression, protein-protein interactions, and post-translational modifications in complex biological samples such as cerebrospinal fluid, serum and plasma, and urine from patients. The publications from these studies have reported greater number of identified proteins, novel biomarker candidates, and post-translational modifications previously unknown. Conclusions: The qualitative and quantitative protein analysis of the protein population of wound tissues or fluids at different stages is important in wound healing research. Given the complexities and analytical challenges of these samples, MS-based proteomic workflows further improved with recent advances offer a powerful and attractive technology for this purpose.

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“…Even before the dramatic growth of biopharmaceutical research, 2D-LC was used extensively for separations of peptides in proteomics research [15]. Now, this prior knowledge in the literature is being applied and extended as researchers use 2D-LC to detect and understand small differences between therapeutic proteins.…”

Section: Peptide Level Analysismentioning

confidence: 99%

The growing role of two-dimensional LC in the biopharmaceutical industry

Wang¹,

Buckenmaier²,

Stoll³

2017

J Appl Bioanal

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IntroductionOver the past two decades, three major trends have been observed in the bioanalysis world.• First, in liquid chromatography (LC) two-dimensional liquid chromatography (2D-LC) has emerged as one of the most active areas of technology advancement [1][2][3]. In 2D-LC, a conventional separation is carried out in the first dimension and aliquots of the effluent are collected and injected to a second-dimension column that has very different separation selectivity compared to the first-dimension column. Therefore, much higher peak capacity, and thus resolving power, can be achieved in 2D-LC compared to 1D-LC. This increased resolving power can be used to increase the likelihood of separating a complex mixture, or decrease the time required to fully separate simpler mixtures. In addition, 2D-LC allows the coupling of two completely different separation modes (e.g. reversed-phase and ionic exchange) in one method. This makes it possible to measure multiple attributes of a target analyte in one method instead of two separate methods. Although 2D-LC research has been going on for more than three decades, the speed of innovation and commercialization has accelerated in the last ten years due to efforts at both universities and instrument companies.• Second, Mass Spectrometry (MS) has become an indispensable tool in bioanalysis [4]. The ability of new MS instruments to more accurately characterize large molecules keeps improving. However, several challenges still remain. MS works best with volatile buffers of limited concentration. The ionization suppression effect still occurs when compounds with very different concentrations (e.g. several orders of magnitude) co-elute in chromatographic separations. These challenges to MS make LC separation a critical part of any bioanalysis workflow.• Finally, in the application area, biopharmaceutical research has become the fastest growing area in the pharmaceutical industry. In particular, monoclonal antibodies (mAbs) are currently the most important class of biotherapeutic molecules. As of 2016, seven of the top-ten-selling drugs were biologics, and six of these were mAb related [5]. In particular, the number one drug Humira (adalimumab) had an annual sales of $15.7 Billion dollars in 2016. Due to the large size of these antibodies at about 150 kDa molecular weight, analyzing them is very challenging. It often takes a suite of analytical tools to fully characterize the molecule and ensure good quality control of drug products involving these molecules. These three trends are developing at the same time and at high speed. It is our opinion that the combination of 2D-LC with MS is emerging as an exciting and essential tool for efficient, high quality biopharmaceutical analysis. In this article, we will discuss examples that demonstrate the power of 2D-LC-MS in this application area.

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Recent advances in peptide separation by multidimensional liquid chromatography for proteome analysis

Cited by 147 publications

References 170 publications

Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

Bottom-Up Mass Spectrometry–Based Proteomics as an Investigative Analytical Tool for Discovery and Quantification of Proteins in Biological Samples

The growing role of two-dimensional LC in the biopharmaceutical industry

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