CLPM: A Cross-Linked Peptide Mapping Algorithm for Mass Spectrometric Analysis

BACKGROUND: Protein-protein, protein-DNA and protein-RNA interactions are of central importance in biological systems. Quadrapole Time-of-flight (Q-TOF) mass spectrometry is a sensitive, promising tool for studying these interactions. Combining this technique with chemical crosslinking, it is possible to identify the sites of interactions within these complexes. Due to the complexities of the mass spectrometric data of crosslinked proteins, new software is required to analyze the resulting products of these studies. RESULT: We designed a Cross-Linked Peptide Mapping (CLPM) algorithm which takes advantage of all of the information available in the experiment including the amino acid sequence from each protein, the identity of the crosslinker, the identity of the digesting enzyme, the level of missed cleavage, and possible chemical modifications. The algorithm does in silico digestion and crosslinking, calculates all possible mass values and matches the theoretical data to the actual experimental data provided by the mass spectrometry analysis to identify the crosslinked peptides. CONCLUSION: Identifying peptides by their masses can be an efficient starting point for direct sequence confirmation. The CLPM algorithm provides a powerful tool in identifying these potential interaction sites in combination with chemical crosslinking and mass spectrometry. Through this cost-effective approach, subsequent efforts can quickly focus attention on investigating these specific interaction sites

Atomic Force Microscopy Study of Protein–Protein Interactions in the Cytochrome CYP11A1 (P450scc)-Containing Steroid Hydroxylase System

<p>Abstract</p> <p>Atomic force microscopy (AFM) and photon correlation spectroscopy (PCS) were used for monitoring of the procedure for cytochrome CYP11A1 monomerization in solution without phospholipids. It was shown that the incubation of 100 &#956;M CYP11A1 with 12% Emulgen 913 in 50 mM KP, pH 7.4, for 10 min at <it>T</it> = 22&#176;C leads to dissociation of hemoprotein aggregates to monomers with the monomerization degree of (82 &#177; 4)%. Following the monomerization procedure, CYP11A1 remained functionally active. AFM was employed to detect and visualize the isolated proteins as well as complexes formed between the components of the cytochrome CYP11A1-dependent steroid hydroxylase system. Both Ad and AdR were present in solution as monomers. The typical heights of the monomeric AdR, Ad and CYP11A1 images were measured by AFM and were found to correspond to the sizes 1.6 &#177; 0.2 nm, 1.0 &#177; 0.2 nm and 1.8 &#177; 0.2 nm, respectively. The binary Ad/AdR and AdR/CYP11A1_mon complexes with the heights 2.2 &#177; 0.2 nm and 2.8 &#177; 0.2 nm, respectively, were registered by use of AFM. The Ad/CYP11A1_mon complex formation reaction was kinetically characterized based on optical biosensor data. In addition, the ternary AdR/Ad/CYP11A1 complexes with a typical height of 4 &#177; 1 nm were AFM registered.</p

Detection of hepatitis C virus core protein in serum by atomic force microscopy combined with&nbsp;mass spectrometry

Yuri D Ivanov,1 Anna L Kaysheva,1,2 Pavel A Frantsuzov,1 Tatyana O Pleshakova,1 Nikolay V Krohin,1 Alexander A Izotov,1 Ivan D Shumov,1 Vasiliy F Uchaikin,1 Vladimir A Konev,1 Vadim S Ziborov,1 Alexander I Archakov11Institute of Biomedical Chemistry, 2PostgenTech Ltd, Moscow, RussiaAbstract: A method for detection and identification of core antigen of hepatitis C virus (HCVcoreAg)-containing particles in the serum was proposed, with due account taken of the interactions of proteotypic peptides with Na+, K+, and Cl- ions. The method is based on a combination of reversible biospecific atomic force microscopy (AFM)-fishing and mass spectrometry (MS). AFM-fishing enables concentration, detection, and counting of protein complexes captured on the AFM chip surface, with their subsequent MS identification. Biospecific AFM-fishing of HCVcoreAg-containing particles from serum samples was carried out using AFM chips with immobilized antibodies against HCVcoreAg (HCVcoreAgim). Formation of complexes between anti-HCVcoreAgim and HCVcoreAg-containing particles on the AFM chip surface during the fishing process was demonstrated. These complexes were registered and counted by AFM. Further MS analysis allowed reliable identification of HCVcoreAg within the complexes formed on the AFM chip surface. It was shown that MS data processing, with account taken of the interactions between HCVcoreAg peptides and Na+, K+ cations, and Cl- anions, allows an increase in the number of peptides identified.Keywords: hepatitis C virus, molecular detector, biospecific fishin