Peptide experimental protocol

FIGURE 15.3 Outline of experimental protocol used for ICAT differential protein expression profiling. Protein mixtures from two cell populations are labeled with light or heavy isotopic versions of a cleavable ICAT reagent. Labeled proteins are combined, subject to multidimensional separation by SCX, RP, and avidin affinity chromatography, then analyzed by tandem MS for peptide and protein identification. Based on the relative ratio of the two isotopically labeled peptides, a relative abundance of protein expression can be determined. 

The introduction of solid-phase synthesis to PMRI-peptides is a result of the systematic and continuous efforts of Verdini and co-workers. 6,51,62,81,84 Unfortunately, these studies are reported only in communications and symposium proceedings, and therefore detailed experimental protocols are not available. Nevertheless, the methodological significance of these studies and their potential applicability justifies a summary in this section. 

Experimental protocols for selective detritylation of peptides containing Cys(Trt) are reported in Section 2.6.6.2.I.3.3. 

In contrast to the observations of Lipton, Kunz and coworkers published in 2005 their critical studies concerning q do-peptide 52 catalysed Strecker reactions and documented difficulties in reaching high levels of enantioselectivities, although the experimental protocol was followed exactly as described by Lipton and coworkers. 

This book is divided into three parts. The first section contains introductory chapters on MS and MSI. Chapter I provides an overview of MSI and focuses on current and future trends in the field. The success of a particular MSI experiment depends on the specific MS approach used. Therefore, the second chapter describes the basic principles of mass spectrometry relevant to MSI and includes cross-references to other chapters of this volume for easier navigation. The third chapter reviews the application of MSI to the study of elemental distributions. Following these introductory chapters, there are multiple protocols that describe qualitative and quantitative measurements of endogenous metabolites and xenobiotics as well as their identification and localization. The last section includes protocols for a variety of MSI approaches developed to study peptide and protein distributions. The experimental protocols presented herein encompass most MSI approaches and technologies for samples from a wide range of biological models including plants, invertebrates, and vertebrates. 

Add 1-10 mg of a protein or antibody containing an available thiol group to the particle suspension. Alternatively, add the protein to be coupled to the particle suspension in an amount equal to 1-10 X molar excess over the calculated monolayer for the protein type to be coupled. The optimal amount of protein to be added should be determined experimentally. Creating thiol groups on proteins or peptides may be done from disulfides by reduction. Alternatively, a thiolation reagent may be used to add thiols to the protein surface for coupling (see the protocols in Chapter 1, Section 4.1). 

The following protocols can be used for the isolation and structural characterization of any natural bioactive peptides from the immune system of invertebrates. The different procedures that will be detailed below refer to the identification and primary structure determination of the Drosophila immune-induced peptides (19,20,23,27,30) and of bioactive peptides from the immune system of other Diptera (17,21,24,31). These approaches were also successfully used for the discovery of bioactive peptides from crustaceans, arachnids, and mollusks. These methods should be considered as a guideline and not as the exact procedure to follow (see Note 3). The suggested procedures will be reported following the normal order of execution, (1) induction of the immune response by an experimental infection, (2) collection of the immunocompetent cells (hemocytes), tissues (epithelia, trachea, salivary glands, etc.)... 

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