Peptide sample preparation

Because ESI is less tolerant of sample conditions, sample preparation is even more critical than in MALDI. In addition, the solvent used for the ESI process is extremely important, unlike in MALDI, where the solvent evaporates away before the analysis. Nonvolatile solvents like water or a 1% concentration of sodium-dodecyl-sulfate (SDS) tend to produce poor sprays and a poor or noisy ion current. A sample in 99% water will not spray as well as a sample prepared in 50% methanol. A peptide sample prepared using acetic acid will also produce better results than one using trifluoroacetic acid (TEA) probably due to a counter ion effect. Nonvolatile salts or small molecules will eventually plug the entrance hole to the mass spectrometer and so they should be avoided. 

The methods I- 4 of sample preparation are classics. As a mle they give a high value of blank and some of them take a lot of time. Microwave sample preparation is perspective, more convenient and much more faster procedure than classical mineralization. There are some problems with the combination Cendall-Kolthoff s kinetic method and microwave sample preparation which discussed. The experimental data of different complex organic matrix are demonstrated (food products on fat, peptides, hydrocarbone matrix, urine etc). 

The quantification of kinins in human tissues or body fluids has been limited due to the inherent difficulties in accurately measuring the concentration of ephemeral peptides. Today HPLC-based and RIA/capture-ELA measurements are established to determine kinins in human plasma, liquor or mine. Serine protease inhibitors need to be added to prevent rapid degradation of the kinins in vitro during sample preparation. Kinins and their degradation products have been studied in various biological milieus such as plasma/ serum, urine, joint fluids, kidney, lung and skeletal muscle [2]. Under normal conditions, the concentration of kinins in these compartments is extremely low for... 

Dai, Y., Whittal R.M., and Li, L., Two-layer sample preparation a method for MALDI-MS analysis of complex peptide and protein mixtures, Anal. Chem., 71, 1087, 1999. 

Herraiz, T., Sample preparation and reversed phase-high performance liquid chromatography analysis of food-derived peptides, Analytica Chimica Acta, 352, 119, 1997. 

Wagner, K., Miliotis, T., Marko-Varga, G., Bischoff, R., Unger, K.K. (2002). An automated online multidimensional HPLC system for protein and peptide mapping with integrated sample preparation. Anal. Chem. 74, 809-820. 

An alternative system proved to be both simpler and more user friendly (Unger et al., 2004 Machtejevas et al., 2006). Thus far we have used this configuration to analyze human plasma, sputum, urine, cerebrospinal fluid, and rat plasma. For each particular analysis we set up an analytical system based on a simple but specific strategy (Figure 9.5). The analysis concept is based on an online sample preparation and a two-dimensional LC system preseparating the majority of the matrix components from the analytes that are retained on a RAM-SCX column followed by a solvent switch and transfer of the trapped peptides. The SCX elution used five salt steps created by mixing 20 mM phosphate buffer (pH 2.5) (eluent Al) and 20 mM phosphate buffer with 1.5 M sodium chloride (eluent Bl) in the following proportions 85/15 70/30 65/45 45/55 0/100 with at the constant 0.1 mL/min flow rate. Desorption of the... 

FIGURE 9.6 The peptide and small protein map from a 100 pL human plasma injection. Columns sample preparation SCX RAM analytical column chromolith performance RP-18, 100 x 0.1 mm I.D. Minute fractions were analyzed using MALDI-TOF MS. Fraction numbers correspond to the time scale. Dot size is related to signal intensity. 

In one study by Hood et al., 282 of 1153 identified proteins were identified by at least 2 unique tryptic peptides from FFPE prostate cancer (PCa) tissue.9 According to the gene ontology classification of the proteins identified, -65% of proteins were predicted to be intracellular proteins, while -50% of the total human proteome is predicted to be located in the intracellular compartment. Additionally, 20% of the proteins identified in the PCa tissue were classified as membrane proteins, which is significantly less than the predicted 40% for the human proteome. This relative disparity is not unexpected, considering the Liquid Tissue sample preparation kit lacks specific protocols for membrane protein extraction. The Liquid Tissue method has also been used for proteomics studies of a variety of FFPE tissue samples, including pancreatic tumors,28 squamous cell carcinoma,4 and oral human papillomavirus lesions.27... 

The choice of the appropriate time for the enzymatic cleavage is a critical point of sample preparation. All types of the reference proteinaceous binders (Section 6.2.3) were cleaved from 1 to 24 h at laboratory temperature 20 25 °C. The peptide peaks in the interval 900 2000 Da were registered below 900 Da the peaks of the matrix interfere and above 2000 Da the incompletely cleaved oligopeptides are present which makes interpretation difficult. The best result, i.e. the highest number of peaks, was obtained after digestion for 2 h at laboratory temperature [32]. 

J. Gobom, M. Schuerenberg, M. Mueller, D. Theiss, H. Lehrach, and E. Nordhoff. a-Cyano-4-hydroxycinnamic Acid Affinity Sample Preparation. A Protocol for MALDI-MS Peptide Analysis in Proteomics. Anal. Chem., 73(2001) 434-438. 

E. Nordhoff, M. Schurenberg, G. Thiele, C. Lubbert, K.-D. Kloeppel, D. Theiss, H. Lehrach, and J. Gobom. Sample Preparation Protocols for MALDI-MS of Peptides and Oligonucleotides Using Prestructured Sample Supports. Int. J. Mass Spectrom., 226(2003) 163-180. 

PITC has been used extensively in the sequencing of peptides and proteins and reactions under alkaline conditions with both primary and secondary amino acids. The methods of sample preparation and derivatization follow a stringent procedure which involves many labour-intensive stages. However, the resulting phenylthio-carbamyl-amino acids (PTC-AA s) are very stable, and the timing of the derivatization step is not as critical as when using OPA. 

One inherent property of peptides that interact with membranes is that self-association or even aggregation will interfere with solubilization by organic solvents or micelles. The preparation, purification and sample preparation of extremely hydrophobic (often transmembrane) peptides is nontrivial and has been addressed by only a few papers [74—79]. 

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