Quantitative Analysis of Snake Venoms Using Soluble Polymer-based Isotope Labeling

Abstract

We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms. The SoPIL reagent selectively captures and isolates cysteine-containing peptides, and the subsequent tagged peptides are released and analyzed using nanoflow liquid chromatography-tandem mass spectrometry. The SoPIL strategy was used to quantify venom proteins from two pairs of venomous snakes: Crotalus scutulatus scutulatus type A, C. scutulatus scutulatus type B, Crotalus oreganus helleri, and Bothrops colombiensis. The identification and accurate quantification of proteins in high throughput analysis are essential components of proteomics strategies for biomarker discovery and studying cellular functions and processes (1, 2). Although the technology to measure mRNA expression is more established, the measurement of differential protein expression provides a more direct, accurate, and versatile way to detect global changes in cellular dynamics in health and disease (3). Over the past several years, many quantitative techniques and strategies have been introduced and thoroughly examined. The traditional and frequently used method to investigate differential protein abundances on a large scale between samples from different sources is the staining of proteins separated by two-dimensional (2D) 1 PAGE. This method falls short in its reproducibility and its inability to detect low abundance and hydrophobic proteins (4). Recently label-free approaches primarily based on the use of LC and highly accurate mass spectrometers have been investigated (5, 6). These methods, however, rely heavily on computational software for data treatment.Stable isotopic labeling has remained the most popular method for quantitative proteomics. The introduction of stable isotopes has typically been carried out by (i) chemical derivatization of proteins or peptides, e.g. via the ICAT (7), isobaric tagging for relative and absolute quantitation (iTRAQ) (8), and isotope-coded protein labeling (9) methods; (ii) enzyme catalyzed labeling, e.g. proteolysis in heavy water incorporates 18 O into the newly generated carboxyl-terminal carboxyl groups (10); and (iii) metabolic labeling methods to incorporate isotopically labeled amino acid residues into proteins (stable isotope labeling using amino acids in cell culture (SI-LAC)) (11). ICAT is probably the best described chemical approach. Over the years, both advantages and disadvantages of ICAT have been recognized. Several variations and modifications of ICAT have been attempted to make it more practical and simpler, such as acid-cleavable ICAT reagents (12) and a solid phase based version (13). The adaptation of the solid phase capture and release process is a significant improvement, and the method obviates extra purification steps and has the potential for automation and high throughput experiments. A side-by-side comparison w...

Identification of Cross-Linked Peptides after Click-Based Enrichment Using Sequential Collision-Induced Dissociation and Electron Transfer Dissociation Tandem Mass Spectrometry

“…Click Labeling and Affinity Enrichment. Procedures by Speers et al and Galan et al 20,21 were modified for click labeling of the enrichment reagent. Briefly, the peptide mixture produced from the in-solution digestion was reduced to dryness and reconstituted in PBS buffer (100 mM sodium phosphate, 0.15 M NaCl, pH 7.5) that was supplemented with 1 µL of 100 mM Tris-[1-benzyl-1H-1,2,3-triazol-4-yl)methylamine (TTA) (0.25 mM final concentration), 20 µL of 50 mM CuSO 4 (2.5 mM final concentration), and 40 µL of 50 mM Tris (2-carboxyethyl) phosphine (5 mM final concentration) to a final volume of 400 µL.…”

Cysteine tagging for MS‐based proteomics

“…SoPIL was applied to the quantitative analysis of snake venoms (see Section IV.B) (Galan et al, 2008). Guttman and co-workers recently reported the fluorescent isotope-coded affinity tag (FCAT) (Rivera-Monroy, Bonn, & Guttman, 2008).…”

“…In addition, a better understanding of geographical and environmental variations in venoms is essential to the development of agents to treat snakebites. Using the SoPIL strategy, Galan and co-workers performed a quantitative analysis of venoms from two pairs of snakes: two Mohave rattlesnakes (types A and B) and two from geographically unrelated snakes (Southern Pacific rattlesnake and Mapanare snake) (Galan et al, 2008). The authors highlighted differences in cysteinerich proteins that revealed intraspecies and geographicalenvironmental variations.…”

Protein-species quantitative venomics: looking through a crystal ball

“…These peptide-centric approaches are mainly used to quantify relative differences in peak intensity of the same analyte between multiple samples. Applications to venomics has been so far scarce, including the relative quantification of type A and type B venoms from the same species of C. s. scutulatus and the venoms from two geographically unrelated snakes from North and South America, C. o. helleri and B. colombiensis , respectively [ 25 ]. More recently, the comparative analyses of venom during the neonate-to-adult transition of Bothrops jararaca [ 26 ] and Gloydius brevicaudus were carried out [ 27 ].…”