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Purpose: Erythrocyte concentrates (ECs) represent the most transfused labile blood products. They are stored at 4°C in additive solutions for up to 56 days. Protein oxidation is a marker of oxidative stress and cysteine residues, whose oxidations are required for physiological cell functions, are highly prone to such modification., Experimental Design: Five ECs from independent donations were followed. Soluble protein extracts were prepared at days 6, 27, and 41, and cysteines were alkylated, reduced, and labeled with infrared dyes. Samples were mixed two by two (day 6 as reference) and analyzed by 2D-DIGE. Detection of labeled cysteines allows quantitative comparison of oxidative status. Spots of interest were analyzed by proteomics., Results: Thirty-two spots containing 43 proteins were classified as increasing, decreasing, or exhibiting a peak of expression during storage. Proteins having catalytic and antioxidant activities were particularly affected during storage, for example, peroxiredoxin-1 and DJ-1 were reversibly oxidized and catalase was irreversibly oxidized. These proteins could be used to evaluate different storage strategies to maintain proper protein function during the overall storage period., Conclusions and Clinical Relevance: This redox-DIGE approach brings new quantitative data on oxidized proteins in stored red blood cells. As previously reported on carbonylation, the oxidative damages differently affect protein functions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Transfusion of red blood cells (RBCs) is a daily medical procedure. Erythrocyte concentrates (ECs) can be stored up to 56 days at 4 °C in saline additive solution mainly composed of adenine and sugar. Such nonphysiological conditions induce the occurrence of storage lesions, such as alterations of metabolism, protein oxidation, and deterioration of rheological properties. Their accumulation tends to decrease the main EC therapeutic property, that is, the oxygenation capacity. Protein carbonylation is a marker of oxidative stress and aging, and its occurrence during RBC storage was earlier characterized as a time-dependent and cellular compartment dependent modification., Experimental Design: Three ECs from independent donations were followed. The carbolynome was here characterized in soluble and membrane extracts (n-dodecyl β-D-maltoside-based extraction buffer) of RBCs stored for 6, 27, and 41 days, through biotin hydrazide derivatization, biotin-avidin affinity purification, SDS-PAGE separation, and LC-MS/MS analyses., Results: A total of 142 and 20 proteins were identified as carbonylated in soluble and membrane extracts, respectively. Particularly, a time-dependent evolution of 26.8% of the soluble carbonylome was observed., Conclusions and Clinical Relevance: Affected cellular mechanisms involve antioxidant defenses, metabolism pathways, and proteasomal degradation. To better store RBCs those functions have to be preserved, which opens new routes of investigation in transfusion medicine., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Electrokinetic supercharging has been integrated in CZE for the development of a highly sensitive methodology for protein tryptic digest analysis. A careful choice of the experimental conditions led to sensitivity enhancement factors between 1000 and 10,000 whilst maintaining a satisfactory resolution. Peptides in the low nanomolar concentration range have been detected despite the use of the poorly sensitive UV absorbance detection mode. The buffer system used in this study is fully suitable for coupling CE to MS.

Protein oxidation mechanisms result in a wide array of modifications, from backbone cleavage or protein crosslinking to more subtle modifications such as side chain oxidations. Protein oxidation occurs as part of normal regulatory processes, as a defence mechanism against oxidative stress, or as a deleterious processes when antioxidant defences are overcome. Because blood is continually exposed to reactive oxygen and nitrogen species, blood proteomics should inherently adopt redox proteomic strategies. In this review, we recall the biochemical basis of protein oxidation, review the proteomic methodologies applied to analyse redox modifications, and highlight some physiological and in vitro responses to oxidative stress of various blood components., (Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Currently two types of chip systems are used in conjunction with MS: out-of-plane devices, where hundreds of nozzles, nanospray emitters are integrated onto a single silicon substrate from which electrospray is established perpendicular to the substrate, and planar microchips, embedding a microchannel at the end of which electrospray is generated in-plane, on the edge of the microchip. In the last two years, carbohydrate research greatly benefited from the introduction and implementation of the chip-based MS. In two laboratories the advantages of the chip electrospray in terms of ionization efficiency, sensitivity, reproducibility, quality of data in combination with high mass accuracy, and resolution of detection were systematically explored for several carbohydrate classes: O- and N-glycopeptides, oligosaccharides, gangliosides and glycoprotein-derived O- and N-glycans, and glycopeptides. The current state-of-the-art in interfacing the chip electrospray devices to high-performance MS for carbohydrate analysis, and the particular requirements for method optimization in both positive and negative ion modes are reviewed here. The recent applications of these miniaturized devices and their general potential for glycomic-based surveys are highlighted.

Despite the consolidation of the specialized proteomics literature around a few established journals, such as Proteomics, Molecular and Cellular Proteomics, and the Journal of Proteome Research, a lot of information is still spread in many different publications from different fields, such as analytical sciences, MS, bioinformatics, etc. The purpose of SPS' Digest is to gather a selection of proteomics articles, to categorize them, and to make the list available on a periodic basis through a web page and email alerts.

The Off-Gel technology was recently described for protein fractionation in a solution placed on top of an immobilized pH gradient gel. In addition, this process was found to remove salts from the biological samples to analyze. This desalting effect is studied experimentally in a conductometric prototype cell. A simplified analytical model is developed to understand this process and a good agreement is found with the conductivity measurements. To illustrate the desalting of a biological sample, a 1 mg.mL(-1) solution of beta-lactoglobulin A in 0.1 M NaCl is subjected to electrophoresis in a single compartment Off-Gel cell. The analysis of the resulting sample by ESI-MS demonstrates the effective removal of salt. A finite element diffusion-migration model is also used to illustrate how the nonuniformity of the electric field in the cell, associated with the salt migration, can slow down the desalting process.

The electrochemically induced mass spectrometric tagging of cysteines by substituted hydroquinones was studied for peptides in a classical electrospray solvent (i.e., MeOH/H2O/AcOH 50/49/1). The tagging efficiency was tested with different hydroquinone compounds on an undecapeptide containing one cysteine residue. 2-carboxymethylhydroquinone was the most reactive probe and revealed to be suitable for cysteine quantification in peptides containing up to three cysteine residues, even in the case of two consecutive cysteines in the sequence. We demonstrate the compatibility of the on-line electrochemical tagging method for the cysteine content analysis of peptides coming from gel-free protein digestion procedures. The identification of bovine serum albumin and human alpha-lactalbumin digest samples in a peptide mapping strategy was greatly improved by the application of the electrotagging technique as post-column treatment. Indeed, the determination of cysteine content in the tryptic peptides provided powerful information in order to enhance the identification score as well as the discrimination against other protein candidates. The tagging method was then applied to the determination of four proteins in a model mixture.

We present the state-of-the-art in miniaturized sample preparation, immunoassays, one-dimensional and multidimensional analyte separations, and coupling of microdevices with electrospray ionization-mass spectrometry. Hyphenation of these different techniques and their relevance to proteomics will be discussed. In particular, we will show that analytical performances of microfluidic analytical systems are already close to fulfill the requirements for proteomics, and that miniaturization results at the same time in a dramatic increase in analysis throughput. Throughout this review, some examples of analytical operations that cannot be achieved without microfluidics will be emphasized. Finally, conditions for the spreading of microanalytical systems in routine proteomic labs will be discussed.

A simple desalting procedure for the coupling of a polymer microchip injector to mass spectrometry is proposed. The overall process is based on the adsorption of proteins on a poly(vinylidene difluoride) (PVDF) membrane, which are then directly eluted in the spraying solution. This microchip-based approach has been successfully applied to small drugs, peptides and proteins originally diluted in phosphate-buffered saline (PBS). Moreover, when eluting the retained proteins in small volumes, a preconcentration is obtained. The combination of single-use, mass-produceable, low-sample-consumption, easy-to-automate, miniaturized polymer injectors with easy-to-handle solution-exchange membranes makes this system particularly amenable to screening applications.