Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples

Chloupek, Rosanne C.; Hancock, William S.; Marchylo, B. A.; Kirkland, J. J.; Boyes, Barry E.; Snyder, Lloyd R.

doi:10.1016/s0021-9673(94)89009-9

Cited by 82 publications

(53 citation statements)

References 4 publications

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“…All of those factors have been utilized to alter the resolution of peptides [21][22][23][24][25][26], e. g. for peptide mapping applications. We have evaluated the impact of selected factors on the orthogonality of peptide separation in the RP-RP 2D-HPLC system.…”

Section: Investigation Of Rp-rp 2d-hplc Orthogonalitymentioning

confidence: 99%

Two‐dimensional separation of peptides using RP‐RP‐HPLC system with different pH in first and second separation dimensions

Gilár

Olivova

Daly

et al. 2005

J of Separation Science

400

368

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Two-dimensional high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than single-dimensional LC. The most popular 2D-HPLC approach used today for proteomic research combines strong cation exchange and reversed-phase HPLC. We have evaluated an alternative mode for 2D-HPLC of peptides, employing reversed-phase columns in both separation dimensions. The orthogonality of 2D separation was investigated for selected types of RP stationary phases, ion-pairing agents and mobile phase pH. The pH appears to have the most significant impact on the RP-LC separation selectivity; the greatest orthogonality was achieved for the system with C18 columns using pH 10 in the first and pH 2.6 in the second LC dimension. Separation was performed in off-line mode with partial fraction evaporation. The achievable peak capacity in RP-RP-HPLC and overall performance compares favorably to SCX-RP-HPLC and holds promise for proteomic analysis.

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Section: Investigation Of Rp-rp 2d-hplc Orthogonalitymentioning

confidence: 99%

Two‐dimensional separation of peptides using RP‐RP‐HPLC system with different pH in first and second separation dimensions

Gilár

Olivova

Daly

et al. 2005

J of Separation Science

400

368

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“…The sample was then eluted by a 5-95% ACN gradient). Recently, an analytical, chromatographic method for separating A␤1-42 from A␤1-40 was described that employed reverse-phase separation at high temperature (29). As indicated in Fig.…”

Section: Immunoprecipitation Of Newly Synthesized A␤ From the Detergementioning

confidence: 99%

Intracellular Accumulation of Insoluble, Newly Synthesized Aβn-42 in Amyloid Precursor Protein-transfected Cells That Have Been Treated with Aβ1–42

Yang

Chandswangbhuvana

Shu

et al. 1999

Journal of Biological Chemistry

106

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Our early study indicates that intracellular A␤1-42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous A␤1-42 also leads to the accumulation of newly synthesized intracellular A␤. Here we describe that newly synthesized A␤, especially A␤n-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after A␤1-42 treatment. These results suggest that intracellular A␤ may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted A␤1-40, the solid-phase intracellular pathway preferentially produces A␤n-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular A␤ also share the same types of A␤ structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway.The major protein component of amyloid deposits associated with Alzheimer's disease (AD) 1 is a 39 -42-amino acid, selfassembling peptide known as the amyloid A␤ peptide. Although significant progress has been made in our understanding of the proteolytic processing of amyloid precursor protein (APP) and the secretion of soluble amyloid A␤ peptide, the mechanisms for the accumulation of insoluble amyloid deposits and their role in AD pathogenesis remains a matter of speculation. It is clear that at least two pathways for APP processing give rise to fragments bearing A␤ sequences at their amino termini: processing by ␣-secretase, which cleaves within the A␤ sequence, thereby precluding amyloid accumulation, and ␤-secretase processing, which generates carboxyl-terminal APP fragments containing the A␤ sequence. Amyloidogenic, ␤-secretase processing events may take place within several intracellular organelles, including the rough endoplasmic reticulum, trans-Golgi network, and lysosomes (1-7). Further processing of APP within the transmembrane domain by ␥-secretase releases soluble 3-and 4-kDa fragments containing all or part of the A␤ sequence (8). Recent evidence indicates that the familial AD amino acid substitutions within the APP transmembrane domain and presenilin favor the production of A␤1-42 form of A␤, which is preferentially localized within diffuse plaques and senile plaques in AD brain. This suggests that A␤1-42 is more closely associated with AD pathogenesis than shorter A␤ isoforms (9, 10).Biochemical studies of synthetic amyloid peptides have elucidated several important properties regarding their ability to assemble into the amyloid fibrils that characteristically accumulate in AD. Peptides that end at residue 42 aggregate much more rapidly than those ending at residue 39 or 40 (11, 12). The pH optimum for ␤-sheet forma...

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“…Simultaneous variation of temperature and solvent gradients was found to be an effective means of optimizing the separation of peptides and proteins [16,97,98]. The retention of peptides and proteins is usually reduced at elevated temperatures, often with non-linear van't Hoff plots [99 -101].…”

Section: Peptides and Proteinsmentioning

confidence: 99%

Temperature programming in liquid chromatography

Greibrokk¹,

Andersen

2001

J. Sep. Science

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The present state of temperature programming in liquid chromatography, including electrochromatography, is reviewed. A relatively limited number of papers making use of temperature programming as a technique in LC has been published so far, while the various effects of temperature are frequently discussed. As a part of the ongoing trend of miniaturization in chromatography, the active use of temperature as a variable in LC is expected to increase significantly. This paper includes an overview of the effects of temperature on column efficiency, retention, and selectivity, particularly in relation to narrow‐bore columns, instrumental features, and some selected applications.

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Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples

Cited by 82 publications

References 4 publications

Two‐dimensional separation of peptides using RP‐RP‐HPLC system with different pH in first and second separation dimensions

Two‐dimensional separation of peptides using RP‐RP‐HPLC system with different pH in first and second separation dimensions

Intracellular Accumulation of Insoluble, Newly Synthesized Aβn-42 in Amyloid Precursor Protein-transfected Cells That Have Been Treated with Aβ1–42

Temperature programming in liquid chromatography

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