Peptide Selection

4 Recent Applications of the Bottom-Up HX-MS/MS Workflow to Pinpoint the HX Properties of Proteins 

Gross and coworkers used the bottom-up HX-FTD method to pinpoint changes in deuterium uptake for single amides in wild-t5q)e and mutant form of apolipoprotein F4 (Apo F4) [65]. Apo F4, a major risk factor for Alzheimer s and cardiovascular diseases, is prone to aggregation where the oligomerization is promoted by residues in the C-terminal region. By comparison of deuterium levels in individual amide residues in wild-type Apo F4 and a mutant that remains in its monomeric form due to the substitutions of four C-terminal residues, several residues involved in the selfassociation process of Apo F4 were identified. 

HX-FTD was also used to study the interaction of the nuclear receptor peroxisome proliferator-activated receptor y (PPARy) with two small-molecule ligands in a bottom-up HX-ETD study by Griffin 

---

Three endogenous opioids have been identified enkephalins, dynorphins and beta-endorphins. These opioid peptides selectively bind to the seven transmembrane GPCRs delta (8), kappa (k), and mu (p). Although dynorphin binds predominately to the k receptor, P-endorphines and enkephalins bind to p and 8 opioid receptors. It is important to note that the analgesia induced by opioids is mediated predominately throngh the p opioid receptor. In vitro studies have shown a decrease in the immnne function and proliferation following p-endorphin administration in rodents (Ray and Cohn 1999) and that the immunosuppressive effects by P-endorphins are steroid-independent (Berkenbosch et al. 1984 Nelson et al. 2000). 

Both absolute quantitation and relative quantitation of species in mixtures is of interest in some circumstances. Quantitation in a 5-minute analysis can be achieved by addition of an internal standard, ideally the target microorganism grown in special media to incorporate heavy isotopes92-95 and determination of the relative peak heights of pairs of proteins from the analyte and the standard. Isotope-labeled proteins or peptides, selected to match proteins or peptides characteristic of target microorganisms, can also serve as internal standards for isotope ratio measurement. The addition of unmatched proteins or peptides is less reliable for either ESI or MALDI measurements because of unpredictable suppression in the variable mixture. 

Kasicka, V, and Prusik, Z. (1989). Isotachophoretic analysis of peptides — selection of electrolyte systems and determination of purity.. Chromatogr. 470, 209—221. 

Strategies for Host Defense Peptide Selection and Optimization 204... 

In addition to the differences in phospholipid content between microbial and host cell membranes, it has been demonstrated that disparity exists between the transmembrane potentials of both organisms. The transmembrane potential is defined by the proton flux between the inner and outer bilayers of the cytoplasmic membrane and ranges from —90 to —110 mV in normal mammalian cells in contrast to transmembrane potentials of —130 to —150mV for logarithmic phase microbes. The differences in these electrochemical gradients have been postulated to drive the influx of peptides into the cell and thus act as a crucial barrier for defining host defense peptide selectivity. ... 

Chemical Structures and Biological Activities of Non-Peptide Selective Kappa Opioid Ligands... 

Endothelins are a family of vasoactive peptides secreted by endothelial cells. The three major endothelin peptides are all composed of 21 amino acids. Endothelins are the most potent vasoconstrictors known. Contraction of vascular smooth muscle in response to endothelin is associated with an increase in intracellular calcium. Increases in endothelin levels have been reported in patients with vasospastic, hypoxic, and ischemic diseases. The two identified isoforms of endothelin receptors have differing affinity for the three endothelin peptides. Selective and nonselective endothelin receptor antagonists are in development for potential use in the treatment of hypertension and other disorders associated with increased vascular resistance. 

The ability to detect Cu2+, Cd2+ and Pb2+ with different electrodes modified in the same way but with different peptides selective for different metals opens the door to fabricating an electrode array for more than one metal ion. To explore that possibility an electrode array with four elements was employed where each electrode was modified with thioctic acid. The four elements were electrodes modified with (a) no peptide (that is thioctic acid alone), (b) Gly-Gly-His, (c) GSH and (d) human angiotensin I (see Fig. 10.5). The electrode array was calibrated by preparing 16 different cocktails of Cu2+, Cd2+ and Pb2+ according to a central composite design [53]. OSWV for each electrode for one of the calibration solutions (0.100 pM Cu2+, 3.00 pM Cd2+ and 0.600 pM Pb2+ in 50 mM ammonium acetate at pH 7.0) are shown in Fig. 10.6. The two features apparent from Fig. 10.6 are that there are only two main peaks due to the overlap of the electrochemistry from... 

Chymotrypsin cleaves a peptide selectively at the carboxyl group of amino acids that have aromatic side chains. The side chain of phenylalanine is a benzyl group, C6H5CH2—. If the dipeptide isolated after treatment with chymotrypsin contains valine (V) and phenylalanine (F), its sequence must be VF. 

Nucleoproteinoids composed from polynucleotide and basic proteinoid have activity to synthesize peptides selectively 28,59). The mechanism is unknown. This is a way to resolve the genetic coding mechanism. Affinity, for example, hydrophobicity or hydrophilicity, between amino acid and nucleic acid is related to their anticodonic... 

Figure 6.6 RGD peptide selectively enhances doxorubicin delivery by SWNTs and toxicity to integrin aT(33-positive cells, (a) Schematic structure of PL-SWNT-RGD-DOX, that is, SWNT functionalized with RGD at the termini of PEG and loaded with doxorubicin on the sidewall by ir-stacking.

---