scholarly journals One-step Isolation of Protein C-terminal Peptides from V8 Protease-digested Proteins by Metal Oxide-based Ligand Exchange Chromatography

2021 ◽  
Author(s):  
Hiroshi Nishida ◽  
Yasushi Ishihama
Keyword(s):  
Metal Oxide ◽  
Ligand Exchange ◽  
Protein C ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Step Method ◽  
C Terminus ◽  
One Step ◽  
Wide Scale ◽  
Internal Peptide

We have developed a one-step method to isolate　protein C-terminal peptides from V8 protease-digested　proteins by metal oxide-based ligand-exchange (MOLEX)　chromatography. V8 protease cleaves the C-terminal　side of Asp and Glu, affording a digested peptide with　two carboxy groups at the C-terminus, whereas the　protein C-terminal peptide has only one α-carboxy　group. In MOLEX chromatography, a stable chelate is　formed between dicarboxylates and metal atoms, so that　the non-terminal (i.e., internal) peptide is retained,　whereas the protein C-terminal peptide flows through the MOLEX column. After optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 μg of peptides (10 μg each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 μg of HeLa peptides was further divided into six fractions by high-pH reversed-phase LC prior to nanoLC/MS/MS, 2202 protein C-termini were identified with less than 3% contamination with internal peptides. We believe this is the largest coverage with the highest purity reported to date in human protein C-terminomics. This fast, simple, sensitive and selective method to isolate protein C-terminal peptides should be useful for profiling protein C-termini on a proteome-wide scale.

scholarly journals Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A

1993 ◽  
Vol 295 (3) ◽  
pp. 813-819 ◽  
Author(s):  
A Varro ◽  
G J Dockray
Keyword(s):  
Ion Exchange ◽  
Secretory Granule ◽  
Secretory Pathway ◽  
Specific Activity ◽  
Brefeldin A ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Gastrin Cells ◽  
Tryptic Fragment ◽  
C Terminus

The precursor for the acid-stimulating hormone gastrin provides a useful model for studies of post-translational processing because defined sites of cleavage, amidation, sulphation and phosphorylation occur within a dodecapeptide sequence. The factors determining these post-translational processing events are still poorly understood. We have used brefeldin A, which disrupts transport from rough endoplasmic reticulum to the Golgi complex, to examine the mechanisms of cleavage, phosphorylation and sulphation of rat progastrin-derived peptides. Biosynthetic products were detected after immunoprecipitation using antibodies specific for the extreme C-terminus of progastrin, followed by reversed-phase and ion-exchange h.p.l.c. Gastrin cells incorporated [3H]tyrosine, [32P]phosphate and [35S]sulphate into both progastrin and its extreme C-terminal tryptic (nona-) peptide. Ion-exchange chromatography resolved four forms of the C-terminal tryptic fragment of progastrin which differed in whether they were phosphorylated at Ser96, sulphated at Tyr103, both or neither. The specific activity of [3H]tyrosine in the peak that was both phosphorylated and sulphated was higher than in the others. Brefeldin A inhibited the appearance of [3H]tyrosine-labelled C-terminal tryptic fragment but there was an accumulation of labelled progastrin and a peptide corresponding to the C-terminal 46 residues of progastrin. Brefeldin A also inhibited incorporation of 32P and 35S into both progastrin and its C-terminal fragment. Thus phosphorylation of Ser96, sulphation of Tyr103 and cleavage at Arg94-Arg95 depend on passage of newly synthesized progastrin along the secretory pathway; as brefeldin A is thought to act proximal to the trans-Golgi, these processing steps would appear to occur distal to this point. The data also indicate that the stores of unphosphorylated C-terminal tryptic fragment are not available for phosphorylation, implying that this modification occurs proximal to the secretory granule; cleavage is known to occur in the secretory granule which suggests that it occurs after phosphorylation.

Preparation and properties of three analogues of [5-leucine]enkephalin, substrates for enzyme conversion studies

1985 ◽  
Vol 50 (6) ◽  
pp. 1329-1334
Author(s):  
Jaroslav Vičar ◽  
Linda Servítová ◽  
Martin Flegel ◽  
Karel Hauzer ◽  
Tomislav Barth
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
Guinea Pig ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Guinea Pig Ileum ◽  
Opioid Activity ◽  
C Terminus ◽  
N Terminus ◽  
Fragment Condensation

Analogues of [5-Leu]enkephalin, prolonged by methionine on the N-terminus or, by lysine or methionine on the C-terminus were prepared by fragment condensation, purified by ion exchange chromatography or high-pressure liquid chromatography. The substances were characterised by their opioid activity in a test on guinea-pig ileum in comparison with the activity of [5-Leu]enkephalin.

Effect of Dynamic Crosslinking on Mechanical Properties of PP/EPDM Blends

1993 ◽  
Vol 58 (11) ◽  
pp. 2642-2650 ◽  
Author(s):  
Zdeněk Kruliš ◽  
Ivan Fortelný ◽  
Josef Kovář
Keyword(s):  
Mechanical Properties ◽  
Impact Strength ◽  
Shear Modulus ◽  
High Impact ◽  
Necessary Condition ◽  
Step Method ◽  
Melt Mixing ◽  
One Step ◽  
High Impact Strength ◽  
Dynamic Curing

The effect of dynamic curing of PP/EPDM blends with sulfur and thiuram disulfide systems on their mechanical properties was studied. The results were interpreted using the knowledge of the formation of phase structure in the blends during their melt mixing. It was shown, that a sufficiently slow curing reaction is necessary if a high impact strength is to be obtained. Only in such case, a fine and homogeneous dispersion of elastomer can be formed, which is the necessary condition for high impact strength of the blend. Using an inhibitor of curing in the system and a one-step method of dynamic curing leads to an increase in impact strength of blends. From the comparison of shear modulus and impact strength values, it follows that, at the stiffness, the dynamically cured blends have higher impact strength than the uncured ones.

Bismuth particles imbedded degradable nanohydrogel prepared by one-step method for tumor dual-mode imaging and chemo-photothermal combined therapy

2019 ◽  
Vol 375 ◽  
pp. 122000 ◽  
Author(s):  
Yang Xuan ◽  
Xian-Lin Song ◽  
Xiao-Quan Yang ◽  
Ruo-Yun Zhang ◽  
Zi-Yu Song ◽  
...  
Keyword(s):  
Combined Therapy ◽  
Dual Mode ◽  
Step Method ◽  
One Step
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