Purification of proteins and peptides for sequence analysis using microcolumn liquid chromatography

Moritz, Robert L.; Simpson, Richard J.

doi:10.1002/mcs.1220040604

Cited by 20 publications

(3 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The reversed-phase capillary column used in this study (150 mm ϫ 0.2-mm inner diameter, Vydac C18) was fabricated as described elsewhere (20,21) and developed with a linear 15-min gradient at 1.6 l/min from 0 to 90% buffer B, where buffer A was 0.1 M aqueous acetic acid, and buffer B was acetonitrile. The electrospray ionization needle was operated at Ϫ4.5 kV.…”

Section: Rp-hplc Analysis Of Ama-1b Trypticmentioning

confidence: 99%

“…The dominant peak was found to correspond to AMA-1B, which was identified by its reactivity on immunoblots with anti-AMA-1B antibodies and by Edman degradation analysis. N-terminal sequence analysis (data not shown) revealed that the signal sequence was cleaved from the mature exported protein between Cys 21 and Ser 22 of the predicted primary translation product.…”

Section: Purification Of Ama-1 Ectodomain (Ama-1b)-recombinantmentioning

confidence: 99%

See 1 more Smart Citation

The Disulfide Bond Structure of Plasmodium Apical Membrane Antigen-1

Hodder

Crewther

Matthew

et al. 1996

Journal of Biological Chemistry

Self Cite

239

207

View full text Add to dashboard Cite

Apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is one of the leading asexual blood stage antigens being considered for inclusion in a malaria vaccine. The ability of this molecule to induce a protective immune response has been shown to be dependent upon a conformation stabilized by disulfide bonds. In this study we have utilized the reversed-phase high performance liquid chromatography of dithiothreitol-reduced and nonreduced tryptic digests of Plasmodium chabaudi AMA-1 secreted from baculovirus-infected insect cells, in conjunction with N-terminal sequencing and electrospray-ionization mass spectrometry, to identify and assign disulfide-linked peptides. All 16 cysteine residues that are conserved in all known sequences of AMA-1 are incorporated into intramolecular disulfide bonds. Six of the eight bonds have been assigned unequivocally, whereas the two unassigned disulfide bonds connect two Cys-Xaa-Cys sequences separated by 14 residues. The eight disulfide bonds fall into three nonoverlapping groups that define three possible subdomains within the AMA-1 ectodomain. Although the pattern of disulfide bonds within subdomain III has not been fully elucidated, one of only two possible linkage patterns closely resembles the cystine knot motif found in growth factors. Sites of amino acid substitutions in AMA-1 that are well separated in the primary sequence are clustered by the disulfide bonds in subdomains II and III. These findings are consistent with the conclusion that these amino acid substitutions are defining conformational disulfide bond-dependent epitopes that are recognized by protective immune responses.

show abstract

Section: Rp-hplc Analysis Of Ama-1b Trypticmentioning

confidence: 99%

Section: Purification Of Ama-1 Ectodomain (Ama-1b)-recombinantmentioning

confidence: 99%

The Disulfide Bond Structure of Plasmodium Apical Membrane Antigen-1

Hodder

Crewther

Matthew

et al. 1996

Journal of Biological Chemistry

Self Cite

239

207

View full text Add to dashboard Cite

show abstract

“…Therefore, column length can be reduced without overly affecting separation and resolution of peptides, and relatively short columns (5-15 cm) can often be used [18]. Therefore, column length can be reduced without overly affecting separation and resolution of peptides, and relatively short columns (5-15 cm) can often be used [18].…”

Section: Packed Bed (Column) Lengthmentioning

confidence: 99%

Peptide Purification by Reversed‐Phase Chromatography

Kusebauch¹,

McBee²,

Bletz³

et al. 2010

Amino Acids, Peptides and Proteins in Organic Chemistry

Self Cite

View full text Add to dashboard Cite

Reversed-phase high-performance liquid chromatography (RP-HPLC) has dominated peptide purification since the late 1970s due to its efficient separation and ease of use with volatile compatible buffer systems for numerous applications [1,2]. RP-HPLC separation of chains of amino acids (peptides and proteins) is based predominantly on reversible hydrophobic interactions between the amino acid side chains with the hydrophobic surface of the chromatographic stationary phase (solid particles or packings) in competition with the flowing mobile phase (chromatographic buffer or solvent). Separation is performed by binding peptides onto the hydrophobic RP stationary phase in polar conditions (e.g., water) and eluting in nonpolar solvents (e.g., organic solvent such as acetonitrile) in the presence of a pH modifying agent. As peptides bind to the stationary phase, the amount of hydrophobic area on the surface of the peptide(s) exposed to the solvent is minimized. Thus, the degree of organized water is decreased with a concomitant favorable increase in entropy of the system. For this reason, it is advantageous, under these solvent conditions, for peptides to associate with the stationary phase upon loading onto the RP-HPLC column. Mobile phase composition is then subsequently modified so that bound peptides are differentially eluted (desorbed) back into the mobile phase. The order of peptide desorption is simply based on their relative hydrophobicity (i.e., least hydrophobic proteins elute first, followed by peptides in increasing order of heir surface hydrophobicity). Peptide elution is usually performed by increasing the organic solvent concentration (either in a stepwise fashion or in a gradient manner). By these means peptides, which are concentrated or trace enriched during the binding and separation process, are eluted in a purified and concentrated form ready for collection or subsequent analysis using hyphenated techniques such as mass spectrometry.

show abstract

Proteomic analysis of colonic crypts from normal, multiple intestinal neoplasia andp53—null mice: A comparison with colonic polyps

Cole

Simpson

2000

Electrophoresis

View full text Add to dashboard Cite

Purification of proteins and peptides for sequence analysis using microcolumn liquid chromatography

Cited by 20 publications

References 9 publications

The Disulfide Bond Structure of Plasmodium Apical Membrane Antigen-1

The Disulfide Bond Structure of Plasmodium Apical Membrane Antigen-1

Peptide Purification by Reversed‐Phase Chromatography

Proteomic analysis of colonic crypts from normal, multiple intestinal neoplasia andp53—null mice: A comparison with colonic polyps

Contact Info

Product

Resources

About