Bidimensional Gel Research Articles

Bidimensional Analyses of the Intra- and Extracellular Proteomes of Steroid Producer Mycobacteria.

The importance of the pathogenic mycobacteria has mainly focused the omic analyses on different aspects of their clinical significance. However, those industrially relevant mycobacteria have received less attention, even though the steroid market sales in 2021 were estimated in $56.45billion.The extracellular proteome, due to its relevance in the sterol processing and uptake, and the intracellular proteome, because of its role in steroids bioconversion, are the core of the present chapter. Both, monodimensional gels, as preparatory analysis, and bidimensional gels as proteome analysis are described. As a proof of concept, the protein extraction methods for both sub-proteomes of Mycobacterium are described. Thus, procedures and relevant key points of these proteome analyses are fully detailed.

Untargeted Proteomics-Based Approach to Investigate Unintended Changes in Genetically Modified Maize for Environmental Risk Assessment Purpose.

Profiling technologies, such as proteomics, allow the simultaneous measurement and comparison of thousands of plant components without prior knowledge of their identity. The combination of these non-targeted methods facilitates a more comprehensive approach than targeted methods and thus provides additional opportunities to identify genotypic changes resulting from genetic modification, including new allergens or toxins. The purpose of this study was to investigate unintended changes in GM Bt maize grown in South Africa. In the present study, we used bi-dimensional gel electrophoresis based on fluorescence staining, coupled with mass spectrometry in order to compare the proteome of the field-grown transgenic hybrid (MON810) and its near-isogenic counterpart. Proteomic data showed that energy metabolism and redox homeostasis were unequally modulated in GM Bt and non-GM maize variety samples. In addition, a potential allergenic protein—pathogenesis related protein −1 has been identified in our sample set. Our data shows that the GM variety is not substantially equivalent to its non-transgenic near-isogenic variety and further studies should be conducted in order to address the biological relevance and the potential risks of such changes. These finding highlight the suitability of unbiased profiling approaches to complement current GMO risk assessment practices worldwide.

The Analysis of Recombination-Dependent Processing of Blocked Replication Forks by Bidimensional Gel Electrophoresis.

The perturbation of the DNA replication process is a threat to genome stability and is an underlying cause of cancer development and numerous human diseases. It has become central to understanding how stressed replication forks are processed to avoid their conversion into fragile and pathological DNA structures. The engineering of replication fork barriers (RFBs) to conditionally induce the arrest of a single replisome at a defined locus has made a tremendous impact in our understanding of replication fork processing. Applying the bidimensional gel electrophoresis (2DGE) technique to those site-specific RFBs allows the visualization of replication intermediates formed in response to replication fork arrest to investigate the mechanisms ensuring replication fork integrity. Here, we describe the 2DGE technique applied to the site-specific RTS1-RFB in Schizosaccharomyces pombe and explain how this approach allows the detection of arrested forks undergoing nascent strands resection.

Optimization of 2-DE and multiplexed detection of O-GlcNAcome, phosphoproteome and whole proteome protocol of synapse-associated proteins within the rat sensorimotor cortex

BackgroundSeveral studies have shown the importance of phosphorylation, O-GlcNAcylation and their interplay in neuronal processes. New methodTo get understanding about molecular mechanisms of synaptic plasticity, we performed a preparation of synaptic protein-enriched fraction on a small sample of rat sensorimotor cortex. We then optimized a multiplexed proteomic strategy to detect O-GlcNAcylated proteins, phosphoproteins, and the whole proteome within the same bidimensional gel. We compared different protocols (solubilisation buffer, reticulation and composition of the gel, migration buffer) to optimize separating conditions for 2D-gel electrophoresis of synaptic proteins. The O-GlcNAcome was revealed using Click chemistry and the azide–alkyne cycloaddition of a fluorophore on O-GlcNAc moieties. The phosphoproteome was detected by Phospho-Tag staining, while the whole proteome was visualized through SYPRORuby staining. ResultsThis method permitted, after sequential image acquisition, the direct in-gel detection of O-GlcNAcome, phosphoproteome, and whole proteome of synapse-associated proteins. ConclusionThis original method of differential proteomic analysis will permit to identify key markers of synaptic plasticity that are O-GlcNAcylated and/or phosphorylated, and their molecular regulations in neuronal processes.

2136-P: Effects of Cytokine Exposure on the Proteome of Human Islets as Evaluated by Shotgun and 2DE Analysis

Pro-inflammatory cytokines (CYT) lead to several changes in human islet (HI) protein expression, but few data are available on CYT-induced alterations at the whole proteome level, including post-translational modifications (PTM). We used label-free shotgun analysis (SG) and bi-dimensional gel electrophoresis (2DE) to explore how CYT modify the proteome of isolated HI. For this purpose, HI were isolated from the pancreas of 5 nondiabetic organ donors (age: 74±6 years; gender: 3M/2F; BMI: 27.9±3.6 kg/m2) and then cultured for 48h in the presence of 50 U/ml interleukin (IL)-1β + 1000 U/ml interferon (IFN)-γ. Then, 40 or 250 μg protein extracts were used respectively for SG or 2DE (to assess lysine-acetylation) analysis, with the SG mass spectrometry data acquired by nano-liquid chromatography tandem mass spectrometry. SG identified around 3000 proteins, of which 286 were differentially expressed after CYT exposure. Of these, 176 were upregulated and 110 downregulated. Among the former, we found mediators of inflammation, proteins of the ER and the immunoproteasome system and oxidative stress modulators (e.g., CXCL2, CXCL10, PSMB8, PSMB9, PSMB10, SOD2). Among the downregulated proteins, there were cathepsins, antioxidant molecules and enzymes of glycolysis and Krebs’ cycle (e.g., CTSH (a candidate gene for T1D), PRDX2, MDH). Ingenuity Pathway Analysis indicated that several upstream regulators were predicted to be differentially affected. Of them, 118 were presumably activated, including STAT1 and STAT2, NF-κB, JAK1, IRF1, HMGB1 and 36 inhibited, such as ACKR2, TRIM24, MAPK1, SUMO3. Bi-dimensional gel electrophoresis showed 151 spots that were lysine-acetylated, of which 2 were upregulated (PRDX3 and SOD1) and 4 downregulated (GDH, CTSD, ACAD9 and EIF4A1) by CYT. The present study shows novel proteomic changes, including specific PTM, induced in human islets by cytokine treatment that could play a role in beta cell dysfunction and demise and be the target for specific treatments. Disclosure F. Grano: None. F. Ciregia: None. M. Ronci: None. M. Mazzoni: None. L. Overbergh: None. C. De Luca: None. M. Suleiman: None. L. Marselli: None. P. De Simone: None. U. Boggi: None. C. Mathieu: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk A/S, Roche Diabetes Care, Sanofi. Speaker's Bureau; Self; AstraZeneca, Novartis AG, Novo Nordisk A/S, Sanofi. D.L. Eizirik: None. A. Lucacchini: None. L. Giusti: None. P. Marchetti: None. M. Bugliani: None. Funding INNODIA

Deficiency of D-alanyl-D-alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis.

D‐Alanyl‐D‐alanine ligase A (DdlA) catalyses the dimerization of two D‐alanines yielding D‐alanyl‐D‐alanine required for mycobacterial peptidoglycan biosynthesis, and is a promising antimycobacterial drug target. To better understand the roles of DdlA in mycobacteria in vivo, we established a cell model in which DdlA expression was specifically downregulated by ddlA antisense RNA by introducing a 380 bp ddlA fragment into pMind followed by transforming the construct into nonpathogenic Mycobacterium smegmatis. The M. smegmatis cell model was verified by plotting the growth inhibition curves and quantifying endogenous DdlA expression using a polyclonal anti‐DdlA antibody produced from the expressed DdlA. Scanning electron microscopy and transmission electron microscopy were used to investigate mycobacterial morphology. Bidimensional gel electrophoresis and mass spectrometry were used to analyze differentially expressed proteins. Consequently, the successful construction of the M. smegmatis cell model was verified. The morphological investigation of the model indicated that DdlA deficiency led to an increased number of Z rings and a rearrangement of intracellular content, including a clear nucleoid and visible filamentous DNA. Proteomic techniques identified six upregulated and 14 downregulated proteins that interacted with each other to permit cell survival by forming a regulatory network under DdlA deficiency. Finally, our data revealed that DdlA deficiency inhibited cell division in mycobacteria and attenuated the process of carbohydrate catabolism and the pathway of fatty acid anabolism, while maintaining active protein degradation and synthesis. N‐Nitrosodimethylamine (NDMA)‐dependent methanol dehydrogenase (MSMEG_6242) and fumonisin (MSMEG_1419) were identified as potential antimycobacterial drug targets.

Estudio proteómico 2DE-DIGE en plasma sanguíneo de pacientes en etapa infantil con leucemia linfoblástica aguda

En Colombia, durante la última década la leucemia linfoblástica aguda (LLA) ha sido el cáncer con mayor incidencia, siendo más del 40% de las muertes por cáncer en menores de edad atribuidas a esta enfermedad. Entre los factores que influyen en estas cifras, el diagnóstico tardío es tal vez el factor más sensible que afecta de manera negativa el éxito del tratamiento. Esta investigación se centró en el estudio del proteoma plasmático de niños colombianos diagnosticados con LLA tipo B, dada su alta incidencia, en comparación con controles en la búsqueda de proteínas que podrían tener potencialidad a ser clasificadas como biomarcadores de diagnóstico. Ahora bien, en vista de los avances en las herramientas proteómicas y de espectrometría de masas y sabiendo que son una alternativa para abordar la complejidad molecular de enfermedades como el cáncer, utilizamos una aproximación proteómica basada en una separación por electroforesis bidimensional diferencial (2DE-DIGE) con posterior separación por cromatografía líquida acoplada a espectrometría de masas en tándem. Se encontraron 8 proteínas con expresión diferencial en plasma de pacientes con LLA-B, entre las cuales resaltan la serotransferrina, la Alfa-1-antitripsina, la haptoglobina, la α2-glicoproteína de zinc y la complemento C3.

Physical evaluation, morphological and identification of seminal proteins in Santa Ines sheep

SUMMARY This study aimed to identify proteins in the seminal plasma associated with fertility in sheep of Santa Inês in Manaus, AM, using twodimensional electrophoresis techniques associated with mass spectrometry. Semen samples from eight adult sheep were collected by removing an aliquot for the physical and morphological assessments of semen and seminal plasma was subjected to SDS-PAGE profile and two-dimensional electrophoresis. Gels were stained with colloidal Coomassie, scanned and analyzed using ImageMaster 2D Platinum software, version 6.0. The selected individual spots were cut from the master gel, digested with trypsin and subjected to identification by mass spectrometry (MALDITof / Tof). Of the 108 spots detected in the gel, it selected 10 differential spots (based on the distribution thereof in the bidimensional gel and pre-analysis of the 2D ImageMaster Platinum Software) identifying 03 proteins: clusterin, a protein 14-3-3 zeta chain and Ram Seminal versicles 22kDa Protein. The identity of these proteins implies that the components of seminal plasma participate in physiological processes involved in sperm protection, motility and sperm capacitation, all associated with fertility. These proteins need to be better studied to see whether the same could be used as molecular markers of fertility as they were also found in other studies conducted with sheep Santa Ines.

Vacuum Infiltration-Centrifugation Method for Apoplastic Protein Extraction in Grapevine.

The apoplastic fluid moving in the extracellular space external to the plasma membrane provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Here, we describe an optimized vacuum-infiltration-centrifugation method to extract soluble proteins from apoplastic fluid of grapevine leaves. This optimized method allows recovering of the grapevine apoplastic soluble proteins suitable for mono- and bi-dimensional gel electrophoresis for further proteomic analysis in order to elucidate their physiological functions.

Proteomic analysis reveals differentially secreted proteins in the urine from patients with clear cell renal cell carcinoma

ObjectiveThe aim of this study was to evaluate the differentially secreted protein profile in the urine from patients with clear cell renal cell carcinoma (ccRCC) using mass spectrometry–based methods. Urine composition can reflect kidney physiology and can be used to detect markers for renal diseases. Moreover, characterization of the secretome is likely to assist in the investigation of new drugs for biological targets and diagnose the ccRCC at an early stage. Methods and materialsUrine samples from patients were divided according to Fuhrman degree (FI-IV), which was associated with the cellular differentiation as good prognosis (GP) and poor prognosis (PP). Healthy individuals were used as the control group (CG). We used both qualitative and quantitative mass spectrometry–based analyses that involved the following approaches: 1-dimensional gel electrophoresis combined with liquid chromatography mass spectrometry in tandem (1DE LC-MS/MS), in-solution digestion combined with label-free 1-dimensional LC-MSE (1D LC-MSE), and bidimensional gel electrophoresis combined with matrix-assisted laser desorption/ionization time of flight in tandem (2DE MALDI-TOF/TOF) or combined with LC-MS/MS. ResultsAll the strategies allowed the identification of 354 proteins from the CG, GP, and PP groups. Qualitative experiments using 1DE LC-MS/MS analysis detected different protein profiles, and 224 proteins were identified in all groups. The label-free MSE quantitative analysis identified 113 proteins and generated novel information on secreted protein profiles, including 49 up-secreted proteins in the urine from patients with ccRCC and 40 down-secreted proteins related to the CG. Proteins such as kininogen-1, uromodulin, apolipoprotein D, polyubiquitin, and CD59 glycoprotein were down secreted according to the groups CG>GP>PP. In contrast, apolipoprotein A, fibrinogen, and haptoglobin were up secreted in patient groups. The same expression profile observed for kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin was corroborated by 2DE LC-MS/MS or 2DE MALDI-TOF/TOF analyses. These 2 strategies also showed 13 differentially secreted proteins among the 3 groups. ConclusionsThe proteins kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin presented similar quantitative protein profiles according to MSE and 2DE approaches. The latter proteins were up secreted and the former ones were down-regulated. The strategies used proved to be valuable in identifying proteins that were differentially secreted in urine from patients with RCC.

Comparative Proteome of Acetobacter pasteurianus Ab3 During the High Acidity Rice Vinegar Fermentation.

As a traditional Asian food for several centuries, vinegar is known to be produced by acetic acid bacteria. The Acetobacter species is the primary starter for vinegar fermentation and has evolutionarily acquired acetic acid resistance, in which Acetobacter pasteurianus Ab3 is routinely used for industrial production of rice vinegar with a high acidity (9%, w/v). In contrast to the documented short-term and low acetic acid effects on A. pasteurianus, here we investigated the molecular and cellular signatures of long-term and high acetic acid responses by proteomic profiling with bidimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF/MS) analyses. Protein spots of interest were selected based on the threshold ANOVA p value of 0.05 and minimal twofold of differential expression, leading to the identification of 26 proteins that are functionally enriched in oxidoreductase activity, cell membrane, and metabolism. The alterations in protein functioning in respiratory chain and protein denaturation may underlay cellular modifications at the outer membrane. Significantly, we found that at higher acidity fermentation phase, the A. pasteurianus Ab3 cells would adapt to distinct physiological processes from that of an ordinary vinegar fermentation with intermediate acidity, indicating increasing energy requirement and dependency of membrane integrity during the transition of acetic acid production. Together, our study provided new insights into the adaptation mechanisms in A. pasteurianus to high acetic acid environments and yield novel regulators and key pathways during the development of acetic acid resistance.

Evaluation of protein extraction methods for enhanced proteomic analysis of tomato leaves and roots.

Proteomics is an outstanding area in science whose increasing application has advanced to distinct purposes. A crucial aspect to achieve a good proteome resolution is the establishment of a methodology that results in the best quality and wide range representation of total proteins. Another important aspect is that in many studies, limited amounts of tissue and total protein in the tissue to be studied are found, making difficult the analysis. In order to test different parameters, combinations using minimum amount of tissue with 4 protocols for protein extraction from tomato (Solanum lycopersicum L.) leaves and roots were evaluated with special attention to their capacity for removing interferents and achieving suitable resolution in bidimensional gel electrophoresis, as well as satisfactory protein yield. Evaluation of the extraction protocols revealed large protein yield differences obtained for each one. TCA/acetone was shown to be the most efficient protocol, which allowed detection of 211 spots for leaves and 336 for roots using 500 µg of leaf protein and 800 µg of root protein per gel.

Oxidative proteome alterations during skeletal muscle ageing

Sarcopenia corresponds to the degenerative loss of skeletal muscle mass, quality, and strength associated with ageing and leads to a progressive impairment of mobility and quality of life. However, the cellular and molecular mechanisms involved in this process are not completely understood. A hallmark of cellular and tissular ageing is the accumulation of oxidatively modified (carbonylated) proteins, leading to a decreased quality of the cellular proteome that could directly impact on normal cellular functions. Although increased oxidative stress has been reported during skeletal muscle ageing, the oxidized protein targets, also referred as to the ‘oxi-proteome’ or ‘carbonylome’, have not been characterized yet. To better understand the mechanisms by which these damaged proteins build up and potentially affect muscle function, proteins targeted by these modifications have been identified in human rectus abdominis muscle obtained from young and old healthy donors using a bi-dimensional gel electrophoresis-based proteomic approach coupled with immunodetection of carbonylated proteins. Among evidenced protein spots, 17 were found as increased carbonylated in biopsies from old donors comparing to young counterparts. These proteins are involved in key cellular functions such as cellular morphology and transport, muscle contraction and energy metabolism. Importantly, impairment of these pathways has been described in skeletal muscle during ageing. Functional decline of these proteins due to irreversible oxidation may therefore impact directly on the above-mentioned pathways, hence contributing to the generation of the sarcopenic phenotype.

30 Years Old: O-GlcNAc Reaches the Age of Reason - Regulation of Cell Signaling and Metabolism by O-GlcNAcylation.

30 Years Old: O-GlcNAc Reaches the Age of Reason - Regulation of Cell Signaling and Metabolism by O-GlcNAcylation.

Multiplexed Detection of O-GlcNAcome, Phosphoproteome, and Whole Proteome within the Same Gel.

The cellular diversity of proteins results in part from their post-translational modifications. Among all of them, the O-GlcNAcylation is an atypical glycosylation, more similar to phosphorylation than classical glycosylations. Highly dynamic, reversible, and exclusively localized on cytosolic, nuclear, and mitochondrial proteins, O-GlcNAcylation is known to regulate almost all if not all cellular processes. Fundamental for the cell life, O-GlcNAcylation abnormalities are involved in the etiology of several inherited diseases. Assessing to O-GlcNAcylation pattern will permit to get relevant data about the role of O-GlcNAcylation in cell physiology. To get understanding about the role of O-GlcNAcylation, as also considering its interplay with phosphorylation, the O-GlcNAc profiling remains a real challenge for the community of proteomists/glycoproteomists. The development of multiplexed proteomics based on fluorescent detection of proteins permits to go further in the understanding of the proteome complexity. We propose herein a multiplexed proteomic strategy to detect O-GlcNAcylated proteins, phosphoproteins, and the whole proteome within the same bidimensional gel. In particular, we investigated the phosphoproteome through the ProQ Diamond staining, while the whole proteome was visualized through Sypro Ruby staining, or after the labeling of proteins with a T-Dye fluorophore. The O-GlcNAcome was revealed by the way of the Click chemistry and the azide–alkyne cycloaddition of a fluorophore on GlcNAc moieties. This method permits, after sequential image acquisition, the direct in-gel detection of O-GlcNAcome, phosphoproteome, and whole proteome.

Controlling the dynamics of a bidimensional gel above and below its percolation transition

The morphology and the microscopic internal dynamics of a bidimensional gel formed by spontaneous aggregation of gold nanoparticles confined at the water surface are investigated by a suite of techniques, including grazing-incidence x-ray photon correlation spectroscopy (GI-XPCS). The range of concentrations studied spans across the percolation transition for the formation of the gel. The dynamical features observed by GI-XPCS are interpreted in view of the results of microscopic imaging; an intrinsic link between the mechanical modulus and internal dynamics is demonstrated for all the concentrations. Our work presents an example of a transition from a stretched to a compressed correlation function actively controlled by quasistatically varying the relevant thermodynamic variable. Moreover, by applying a model proposed some time ago by Duri and Cipelletti [Europhys. Lett. 76, 972 (2006)] we are able to build a master curve for the shape parameter, whose scaling factor allows us to quantify a "long-time displacement length." This characteristic length is shown to converge, as the concentration is increased, to the "short-time localization length" determined by pseudo-Debye-Waller analysis of the initial contrast. Finally, the intrinsic dynamics of the system is then compared with that induced by means of a delicate mechanical perturbation applied to the interface.

Differential Protein Expression in the Nucleus Accumbens and Amygdala of Lewis and Fischer 344 Rats, and its Relevance in Drug Addiction

The Lewis (LEW) and Fischer 344 (F344) rat strains are widely used as models of genetic vulnerability to drug addiction, par- ticularly since these two strains show different patterns of drug self- administration. We previously performed an unbiased and genome- wide microarray analysis to define the differences in gene expression in the nucleus accumbens (NAcc) and prefrontal cortex between LEW and F344 rats. In this study, we set out to determine the strain differences in the NAcc and amygdala at the protein level using bidimensional gel electrophoresis. As such, 11 proteins were found to be differen- tially expressed between these two strains, the activity of which was mainly related to vesicular trafficking (STXBP1 and GDI1), energy metabolism (ATP5B, ENO2, and ACOT7), and cytoskeletal formation. This information might be useful to search for new molecular targets to screen individuals at high risk of developing an addictive disorder.

Effect of Diluents and Asphaltenes on Interfacial Properties and Steam-Assisted Gravity Drainage Emulsion Stability: Interfacial Rheology and Wettability

In steam-assisted gravity drainage (SAGD) operations, the produced fluids are complex water-in-oil-in-water (W/O/W) emulsions. A diluent is often added to reduce the density and viscosity of the heavy crude oil. However, the quality and composition of the diluents may in some cases increase emulsion stability and cause the dehydration of the oil to be more difficult because there are more surface-active agents added to the oil coming from the diluent streams. Thus, this work was aimed at studying the effect of three different diluents on interfacial film formation of a Canadian heavy oil. Interfacial elasticity and compressibility were evaluated, and the results were then correlated with emulsion stability. The wettability of the systems was also studied. The systems studied behaved as if a bidimensional gel near its gelation point had organized at the interface, in which adsorbed amphiphilic materials, such as asphaltenes and resins, self-aggregated at the interface, forming a network that acts as a stabilizing mechanism for produced emulsions. When the interface was aged, its gel strength was much higher than the fresh interface, suggesting an explanation as to why aged emulsions are more difficult to treat. Unlike elasticity and interfacial tension measurements, it was demonstrated that compressibility measurements can predict emulsion stability under different conditions. The higher the crumpling film ratio and the lower the compressibility, the more stable the emulsion. This test method gives more insights into the mechanisms of emulsion stabilization caused by diluents and asphaltenes and can potentially be employed to study the structure and demulsifying performance relationships of emulsion breakers (EBs) and reverse emulsion breakers (REBs).

Statistical properties and morphology of a 2D gel network at the air/water interface

We report here a detailed characterization of the steps leading to the formation of a bidimensional gel at the air/water interface. The 2D geometry is peculiar in that it allows a continuous and controlled variation of the density of the system over a wide range. The evolution of the statistical properties of the structured network, as a function of the externally controlled density, is related to the corresponding evolution of the mechanical properties. We identified three steps in the gel formation: a first step occurs in the incubation time, in which the individual nm-sized gold nanoparticle aggregate to form quasi 1D structures of typical length of a few microns. The second step occurs in the first stages of the compression, with the growth of the branched structure, finally yielding to the onset of the infinite percolative cluster, which is related to the building of the mechanical elastic modulus. The final step identifies with the homogenization and regularization of the distribution of holes sizes, which relates to the further increase of the elastic modulus upon compression. We speculate that this study should help in the formulation of gels with desired characteristics, such as increased mechanical strength, or increased mobility.

Proteomics applied to transfusion plasma: the beginning of the story

'Safe blood' is and has always been the major concern in transfusion medicine. Plasma can undergo virus inactivation treatments based on physicochemical, photochemical or thermal methodologies for pathogen inactivation. The validation of these treatments is essentially based on clottability assays and clotting factors' titration; however, their impact on plasma proteins at the molecular level has not yet been evaluated. Proteomics appears as particularly adapted to identify, to localize and, consequently, to correlate these modifications to the biological activity change. At the crossroads of biology and analytical sciences, proteomics is the large-scale study of proteins in tissues, physiological fluids or cells at a given moment and in a precise environment. The proteomic strategy is based on a set of methodologies involving separative techniques like mono- and bidimensional gel electrophoresis and chromatography, analytical techniques, especially mass spectrometry, and bioinformatics. Even if plasma has been extensively studied since the very beginning of proteomics, its application to transfusion medicine has just begun. In the first part of this review, we present the principles of proteomics analysis. Then, we propose a state of the art of proteomics applied to plasma analysis. Finally, the use of proteomics for the evaluation of the impact of storage conditions and pathogen inactivation treatments applied to transfusion plasma and for the evaluation of therapeutic protein fractionated is discussed.