High-Performance Liquid Chromatography−Electrospray Ionization Mass Spectrometry Using Monolithic Capillary Columns for Proteomic Studies

Premstaller, Andreas; Oberacher, Herbert; Walcher, W; Timperio, Anna Maria; Zolla, Lello; Chervet, § Jean-Pierre; Cavusoglu, Nükhet; Dorsselaer, and Alain van; Huber, Christian G.

doi:10.1021/ac010046q

Cited by 206 publications

(158 citation statements)

References 40 publications

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“…The original analytical scale size columns were supplemented with their capillary counterparts in the early 2000s [5,[12][13][14][15][16][17][18][19]. These sub-millimeter internal diameter columns facilitate direct splitless coupling with electrospray ionization mass spectrometry (ESI-MS) [20][21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Stability and repeatability of capillary columns based on porous monoliths of poly(butyl methacrylate-co-ethylene dimethacrylate)

Geiser

Eeltink

Švec

et al. 2007

Journal of Chromatography A

109

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Monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) capillary columns have been prepared via either thermally or photochemically initiated polymerization of the corresponding monomers and the repeatability of their preparation has been explored. Three separate batches of five columns each were prepared using thermal and photochemical initiation for a total of thirty columns. All thirty capillary columns were tested in liquid chromatography-electrospray ionizationmass spectrometry mode for the separation of a model mixture of three proteins -ribonuclease A, cytochrome c and myoglobin. Excellent repeatability of retention times was observed for the proteins as evidenced by relative standard deviation (RSD) values of less than 1.5%. Somewhat broader variations with RSD values of up to 10% were observed for the pressure drop in the columns. The stability of retention times was also monitored using a single monolithic column and no significant shifts in either retention times or back pressure was observed in a series of almost 2200 consecutive protein separations.

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Section: Introductionmentioning

confidence: 99%

Stability and repeatability of capillary columns based on porous monoliths of poly(butyl methacrylate-co-ethylene dimethacrylate)

Geiser

Eeltink

Švec

et al. 2007

Journal of Chromatography A

109

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“…By virtue of its high resolving power and the utilization of eluents that are compatible with ESI-MS (28), RP-HPLC has become the separation method of choice for direct interfacing with ESI-MS (29,30). In this article, the strategy of RP-HPLC-ESI-MS has been used to separate and identify isomeric forms of major and minor antenna proteins in different plant species.…”

mentioning

confidence: 99%

Isoforms of Photosystem II Antenna Proteins in Different Plant Species Revealed by Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Huber¹,

Timperio²,

Zolla³

2001

Journal of Biological Chemistry

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The high selectivity offered by reversed-phase highperformance liquid chromatography on-line coupled to electrospray ionization mass spectrometry has been utilized to characterize the major and minor light-harvesting proteins of photosystem II (Lhcb). Isomeric forms of the proteins, revealed either on the basis of different hydrophobicity enabling their chromatographic separation or on the basis of different molecular masses identified within one single chromatographic peak, were readily identified in a number of monocot and dicot species. The presence of several Lhcb1 isoforms (preferably in dicots) can explain the tendency of dicot Lhcb1 to form trimeric aggregates. The Lhcb1 molecular masses ranged from 24,680 to 25,014 among different species, whereas within the same species, the isoforms differed by 14 -280 mass units. All Lhcb1 proteins appear to be highly conserved among different species such that they belong to a single gene group that has several different gene family members. In all species examined, the number of isoforms corresponded more or less to the genes cloned previously. Two isoforms of Lhcb3 were found in petunia and tomato. For Lhcb6, the most divergent of all light-harvesting proteins, the greatest number of isoforms was found in petunia, tobacco, tomato, and rice. Lhcb2, Lhcb4, and Lhcb5 were present in only one form. The isoforms are assumed to play an important role in the adaptation of plants to environmental changes. Photosynthesis in higher plants begins with the absorption of light by the light-harvesting complex of photosystem II (LH-CII)1 through two types of light-harvesting proteins: the major antenna, comprising the proteins Lhcb1, Lhcb2, and Lhcb3, and the minor antenna, comprising the proteins Lhcb4, Lhcb5, and Lhcb6. Although the LHCII has been extensively studied since its discovery more than 35 years ago, there are still several questions about the number of polypeptides it contains and its supramolecular organization. It is known that LHCII apoproteins are encoded by families of nuclear genes (1-3) and over 40 nucleotide sequences encoding LHCII proteins from more than 15 different plant species have been cloned and sequenced (4). Moreover, a large number of protein bands can be resolved by gel electrophoresis from LHCII preparations (3). The precise relationship between the multiple functional roles played by LHCII, the diversity of polypeptides found in the membrane, and the complexity of the gene family that encodes the LHCII apoproteins has not yet been fully established. The observed protein heterogeneity has, in fact, a number of possible origins, thus the different protein isoforms could be: (i) products of distinct genes (5), (ii) post-translational modifications of one or a few primary translation products (6 -8), (iii) cleavage products of a common precursor at successive maturation stages (9), or (iv) artifacts introduced during sample workup and analysis. The antenna system is organized into a mobile complex containing homotrimers of Lhcb1 and heterotrimers co...

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“…Recently introduced monolithic-columns [63] operate at significantly higher flow rates than classical HPLC-columns at slightly lower capacities. Thereby, faster separations with better resolution are achievable due to reduced peak-broadening.…”

Section: Localization Of Phosphorylation Sitesmentioning

confidence: 99%

State-of-the-art in phosphoproteomics

2005

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Presently, phosphorylation of proteins is the most studied and best understood PTM. However, the analysis of phosphoproteins and phosphopeptides is still one of the most challenging tasks in contemporary proteome research. Since not every phosphoprotein is accessible by a certain method and identification of the phosphorylated amino acid residue is required in the majority of cases, various strategies for the detection and localization of phosphorylations have been developed. Identification and localization of protein phosphorylations is mostly done by MS nowadays but phosphoproteins and -peptides are often suppressed in comparison to the unphosphorylated species if measured in complex mixtures. Thus, the isolation of pure phosphopeptide samples is a main task. This review gives an overview over the most frequently used methods in isolation and detection of phosphoproteins and -peptides such as specific enrichment or separation strategies as well as the localization of the phosphorylated residues by various mass spectrometric techniques.

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High-Performance Liquid Chromatography−Electrospray Ionization Mass Spectrometry Using Monolithic Capillary Columns for Proteomic Studies

Cited by 206 publications

References 40 publications

Stability and repeatability of capillary columns based on porous monoliths of poly(butyl methacrylate-co-ethylene dimethacrylate)

Stability and repeatability of capillary columns based on porous monoliths of poly(butyl methacrylate-co-ethylene dimethacrylate)

Isoforms of Photosystem II Antenna Proteins in Different Plant Species Revealed by Liquid Chromatography-Electrospray Ionization Mass Spectrometry

State-of-the-art in phosphoproteomics

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