Performance of a novel sieving matrix of poly(vinyl alcohol)/acrylamide copolymer in electrophoretic separations of high molecular weight proteins from red cell membrane.

The analysis of high molecular weight (HMW) proteins from complex mixtures is still a challenge in proteomics. This work introduces a novel hydrogel obtained by the copolymerization of an allyl-PVA derivative with acrylamide and bisacrylamide and applies this matrix to the electrophoretic separation of HMW proteins. By inducing gelation of polyacrylamide in the presence of variable amounts of allyl-PVA, it is possible to control and vary the average gel porosity. This gel is easy to produce and handle and offers the advantage of being highly mechanically resistant and macroporous. The new matrix was tested in mono-dimensional separations of complex protein mixtures extracted from red cell membranes with different detergents. The improved performance of this macroporous matrix allowed to identify new proteins by MS and immunoblot analysis using specific antibodies. In particular, the resolution of proteins ranging in size between 97 and 279 kDa was greatly improved here compared to standard polyacrylamide gels, suggesting that this matrix can be a useful tool in routine analysis of HMW proteins in cell biology.

In Humanized Sickle Cell Mice, Imatinib Protects Against Sickle Cell-Related Injury.

Drug repurposing is a valuable strategy for rare diseases. Sickle cell disease (SCD) is a rare hereditary hemolytic anemia accompanied by acute and chronic painful episodes, most often in the context of vaso-occlusive crisis (VOC). Although progress in the knowledge of pathophysiology of SCD have allowed the development of new therapeutic options, a large fraction of patients still exhibits unmet therapeutic needs, with persistence of VOCs and chronic disease progression. Here, we show that imatinib, an oral tyrosine kinase inhibitor developed for the treatment of chronic myelogenous leukemia, acts as multimodal therapy targeting signal transduction pathways involved in the pathogenesis of both anemia and inflammatory vasculopathy of humanized murine model for SCD. In addition, imatinib inhibits the platelet-derived growth factor-B-dependent pathway, interfering with the profibrotic response to hypoxia/reperfusion injury, used to mimic acute VOCs. Our data indicate that imatinib might be considered as possible new therapeutic tool for chronic treatment of SCD.

Life and Death of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficient Erythrocytes - Role of Redox Stress and Band 3 Modifications.

SUMMARY: G6PD catalyzes the first, pace-making reaction of pentosephosphate cycle (PPC) which produces NADPH. NADPH maintains glutathione and thiol groups of proteins and enzymes in the reduced state which is essential for protection against oxidative stress. Individuals affected by G6PD deficiency are unable to regenerate reduced glutathione (GSH) and are undefended against oxidative stress. G6PD deficiency accelerates normal senescence and enhances the precocious removal of chronologically young, yet biologically old cells. The term hemolytic anemia is misleading because RBCs do not lyse but are removed by phagocytosis. Acute hemolysis by fava bean ingestion in G6PD deficient individuals (favism) is described being the best-studied natural model of oxidant damage. It bears strong analogies to hemolysis by oxidant drugs or chemicals. Membrane alterations observed in vivo during favism are superimposable to changes in senescent RBCs. In summary, RBC membranes isolated from favic patients contained elevated amounts of complexes between IgG and the complement fragment C3b/C3c and were prone to vesiculation. Anti-band 3 IgG reacted to aggregated band 3-complement complexes. In favism extensive clustering of band 3 and membrane deposition of hemichromes were also observed. Severely damaged RBCs isolated from early crises had extensive membrane cross-bonding and very low GSH levels and were phagocytosed 10-fold more intensely compared to normal RBCs.

Bcl2-A1 interacts with pro-caspase-3: implications for amyotrophic lateral sclerosis.

Expression of mutant SOD1 typical of familial amyotrophic lateral sclerosis (ALS) induces the expression of Bcl2-A1, a member of the Bcl2 family of proteins, specifically in motor neurons of transgenic mice. In this work, we have used immortalized motor neurons (NSC-34) and transgenic mice expressing mutant SOD1 to unravel the molecular mechanisms and the biological meaning of this up-regulation. We report that up-regulation of Bcl2-A1 by mutant SOD1 is mediated by activation of the redox sensitive transcription factor AP1 and that Bcl2-A1 interacts with pro-caspase-3 via its C-terminal helix α9. Furthermore, Bcl2-A1 inhibits pro-caspase-3 activation in immortalized motor neurons expressing mutant SOD1 and thus induction of Bcl2-A1 in ALS mice represents a pro-survival strategy aimed at counteracting the toxic effects of mutant SOD1. These data provide significant new insights on how molecular signaling, driven by expression of the ALS-causative gene SOD1, affects regulation of apoptosis in motor neurons and thus may have implications for ALS therapy, where prevention of motor neuronal cell death is one of the major aims.

Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in ß-thalassemic erythropoiesis.

BACKGROUND: ß-thalassemic syndromes are inherited red cell disorders characterized by severe ineffective erythropoiesis and increased levels of reactive oxygen species whose contribution to ß-thalassemic anemia is only partially understood. DESIGN AND METHODS: We studied erythroid precursors from normal and ß-thalassemic peripheral CD34(+) cells in two-phase liquid culture by proteomic, reverse transcriptase polymerase chain reaction and immunoblot analyses. We measured intracellular reactive oxygen species, heme levels and the activity of δ-aminolevulinate-synthase-2. We exposed normal cells and K562 cells with silenced peroxiredoxin-2 to H(2)O(2) and generated a recombinant peroxiredoxin-2 for kinetic measurements in the presence of H(2)O(2) or hemin. RESULTS: In ß-thalassemia the increased production of reactive oxygen species was associated with down-regulation of heme oxygenase-1 and biliverdin reductase and up-regulation of peroxiredoxin-2. In agreement with these observations in ß-thalassemic cells we found decreased heme levels related to significantly reduced activity of the first enzyme of the heme pathway, δ-aminolevulinate synthase-2 without differences in its expression. We demonstrated that the activity of recombinant δ-aminolevulinate synthase-2 is inhibited by both reactive oxygen species and hemin as a protective mechanism in ß-thalassemic cells. We then addressed the question of the protective role of peroxiredoxin-2 in erythropoiesis by exposing normal cells to oxidative stress and silencing peroxiredoxin-2 in human erythroleukemia K562 cells. We found that peroxiredoxin-2 expression is up-regulated in response to oxidative stress and required for K562 cells to survive oxidative stress. We then showed that peroxiredoxin-2 binds heme in erythroid precursors with high affinity, suggesting a possible multifunctional cytoprotective role of peroxiredoxin-2 in ß-thalassemia. CONCLUSIONS: In ß-thalassemic erythroid cells the reduction of δ-aminolevulinate synthase-2 activity and the increased expression of peroxiredoxin-2 might represent two novel stress-response protective systems.

LRRK2 Modulates the Exocyst Complex Assembly by Interacting with Sec8.

Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson's disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking, likely by Rab phosphorylation, that in turn may regulate different aspects of neuronal physiology. Here we show that LRRK2 interacts with Sec8, one of eight subunits of the exocyst complex. The exocyst complex is an evolutionarily conserved multisubunit protein complex mainly involved in tethering secretory vesicles to the plasma membrane and implicated in the regulation of multiple biological processes modulated by vesicle trafficking. Interestingly, Rabs and exocyst complex belong to the same protein network. Our experimental evidence indicates that LRRK2 kinase activity or the presence of the LRRK2 kinase domain regulate the assembly of exocyst subunits and that the over-expression of Sec8 significantly rescues the LRRK2 G2019S mutant pathological effect. Our findings strongly suggest an interesting molecular mechanism by which LRRK2 could modulate vesicle trafficking and may have important implications to decode the complex role that LRRK2 plays in neuronal physiology.

Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease.

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for alpha-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.

Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells.

Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS(3) (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.

Immunohistochemical and proteomic profile of melanotic medulloblastoma.

We present the case of a 6-year-old male affected by an infratentorial tumor. Histological diagnosis was melanotic medulloblastoma. Immunohistochemistry showed in the melanin rich areas positive cells for HMB45. We performed a proteomic study to compare protein profiles in melanotic versus non-melanotic areas. Protein profiles of different areas of the tumor displayed similarity, with the exception of seven proteins. In accordance with the hypothesis that melanotic medulloblastomas produce oculo-cutaneous melanin, proteomic analysis showed melanocytic-associated antigens and epidermal autoantigen 450K in the pigmented nodule; both these proteins have a significant role as markers of melanotic elements.

Bitopertin, a selective oral GLYT1 inhibitor, improves anemia in a mouse model of ß-thalassemia.

Anemia of ß-thalassemia is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of ß-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage, as indicated by a reduction in membrane-associated free α-globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes, and in heme-regulated inhibitor activation and eIF2α phosphorylation. The improvement of ß-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation and Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of ß-thalassemia.

PTPepsilon has a critical role in signaling transduction pathways and phosphoprotein network topology in red cells.

Protein tyrosine phosphatases (PTPs) are crucial components of cellular signal transduction pathways. Here, we report that red blood cells (RBCs) from mice lacking PTPepsilon (Ptpre(-/-)) exhibit (i) abnormal morphology; (ii) increased Ca(2+)-activated-K(+) channel activity, which was partially blocked by the Src family kinases (SFKs) inhibitor PP1; and (iii) market perturbation of the RBC membrane tyrosine (Tyr-) phosphoproteome, indicating an alteration of RBC signal transduction pathways. Using the signaling network computational analysis of the Tyr-phosphoproteomic data, we identified seven topological clusters. We studied cluster 1 containing Fyn, SFK, and Syk another tyrosine kinase. In Ptpre(-/-)mouse RBCs, the activity of Fyn was increased while Syk kinase activity was decreased compared to wild-type RBCs, validating the network computational analysis, and indicating a novel signaling pathway, which involves Fyn and Syk in regulation of red cell morphology.

Naturally occurring anti-band 3 antibodies and red blood cell removal under physiological and pathological conditions.

Naturally occurring antibodies (NAbs) directed to band 3 protein (major erythrocyte membrane protein) are involved in the clearance of red blood cell (RBC) at the end of their lifespan as well as in the removal of RBC in different hereditary haemolytic disorders and in malaria. In all cited situations RBC undergoes oxidative stress and hemichromes (haemoglobin degradation products) are formed. Hemichromes possess a strong affinity for band 3 cytoplasmic domain and, following their binding, lead to band 3 oxidation and clusterisation. Those band 3 clusters show increased affinity for NAbs which activate complement and finally trigger the phagocytosis of altered RBC. During intra-erythrocytic malaria parasite growth, NAbs begin to bind to RBC surface at early parasite development stages increasing their abundance in parallel with parasite development. Interestingly, a number of hereditary haemolytic disorders, known to exert a protective effect on malaria, tend to exacerbate this phenomenon leading to a more precocious and effective opsonization of diseased RBC infected by malaria parasites. The exact definition of band 3 neo-antigens and the mechanism of their surface exposure are still unclear. Also band 3 clusterisation is only superficially understood, new insights about band 3 phosphorylation by Src kinases suggest the presence of a complex regulatory pathway.

Proteomic identification of heat shock protein 27 as a differentiation and prognostic marker in neuroblastoma but not in Ewing's sarcoma.

Neuroblastoma (NB) and Ewing's sarcoma (ES) cell lines were analysed by two-dimensional gel electrophoresis (2-DE) searching for new diagnostic/prognostic markers. Protein expression profiles displayed a high degree of similarity with the exception of marked heat shock protein (HSP) 27 and less marked HSP60 and HSP70 family up-modulations in NB cells. HSP27, which showed peculiar variability in different NB cell preparations, responded to all trans-retinoic acid treatment in NB cells but not in ES cells at gene and protein expression levels. Immunohistochemistry studies showed different behaviours of HSP27 and HSP70 expression in NB and ES biopsies. HSP27 was less expressed, whereas HSP70 was more expressed in the immature areas of NB. HSP27 expression showed positive and statistically significant correlation with favourable prognosis, and HSP27 expression also negatively correlated with increasing aggressiveness of histological type. In ES, both chaperones were expressed without characteristic patterns. Our results suggest that HSP27, after further clinical validations, could be used as a marker of neuronal differentiation in vivo for the assessment of the biological behaviour of NB and for the risk stratification of patients.

Highly specific detection of prostate-specific antigen-positive cells in the blood of patients with prostate cancer or benign prostatic hyperplasia, using a real-time reverse-transcription-polymerase chain reaction method with improved sensitivity.

OBJECTIVE: To assess the presence of circulating prostate-specific antigen (PSA)-expressing cells in patients with prostate cancer or benign prostatic hyperplasia (BPH), and to determine their diagnostic usefulness using a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) method. PATIENTS, SUBJECTS AND METHODS: Venous blood samples were obtained from 175 patients with prostate cancer (12 metastatic and 163 not metastatic), 49 with BPH, and 50 healthy volunteers. To improve the specificity and sensitivity of the qRT-PCR three innovative features were combined; a primer overlapping two adjacent exons to inhibit nonspecific amplification; a no-end-point first round amplification to increase the sensitivity; and a target-specific primer for the RT phase to increase the specificity. RESULTS: The sensitivity of the method was 1 cell/mL of blood and the interassay coefficient of variation was 10.5%. None of the healthy subjects tested positively, while 9% of those with prostatic cancer and 14% with BPH had PSA-positive cells in the blood. There was a positive association between a positive test and the National Comprehensive Cancer Network classification in the patients with newly diagnosed prostate cancer (P = 0.022). There were no additional statistically significant associations. CONCLUSION: Our results strongly indicate that although there were no false-positive results and the sensitivity of the method was increased to maximal levels, a low frequency of positive results in patients with prostatic cancer and a high frequency of positive results in those with BPH seems to discourage the use of PSA-positive circulating cells in the search for a clinical diagnosis of prostate cancer.

Inhibition of heat shock proteins (HSP) expression by quercetin and differential doxorubicin sensitization in neuroblastoma and Ewing's sarcoma cell lines.

Neuroblastoma (NB) and Ewing's sarcoma (ES) represent the most common extracranial solid tumors of childhood. Heat shock proteins (HSP) are elevated in cancer cells and their over-expression was correlated to drug-resistance. In this work we identified the HSP by a sensitive proteomic analysis of NB and ES cell lines, then, we studied the HSP response to doxorubicin. Some identified HSP were constitutively more expressed in NB than in ES cells. Doxorubicin-stimulated HSP response only in NB cells. Quercetin was found to inhibit HSP expression depleting heat shock factor 1 (HSF1) cellular stores. Quercetin caused a higher anti-proliferative effect in NB (IC(50): 6.9 +/- 5.8 mumol/L) than in ES cells (IC(50): 85.5 +/- 53.1 mumol/L). Moreover, quercetin caused a very pronounced doxorubicin sensitizing effect in NB cells (241 fold IC(50) decrease) and a moderate effect in ES cells. HSP involvement in NB cells sensitization was confirmed by the silencing of HSF1. Quercetin treatment and HSF1 silencing increased the pro-apoptotic effect of doxorubicin. In conclusion, the higher HSP levels, observed in NB cells, did not confer increased resistance to doxorubicin; on the contrary, HSP inhibition by quercetin or gene silencing caused higher sensitization to doxorubicin. These results may have a potential application in the treatment of NB.

Analysis of candidate genes through a proteomics-based approach in primary cell lines from malignant melanomas and their metastases.

Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression.

In vivo reduction of erythrocyte oxidant stress in a murine model of beta-thalassemia.

BACKGROUND AND OBJECTIVES: Oxidant damage is an important contributor to the premature destruction of erythrocytes and anemia in thalassemias. To assess the extent of oxidant damage of circulating erythrocytes and the effects of antioxidant therapy on erythrocyte characteristics and anemia, we used a mouse model of human beta-thalassemia intermedia (b1/b2 deletion). DESIGN AND METHODS: Several parameters indicative of oxidant damage were measured at baseline and following administration of the semi-synthetic flavonoid antioxidant, 7-monohydroxyethylrutoside (monoHER), to beta-thalassemic mice at a dose of either 500 mg/kg i.p. once a day (n=6) or 250 mg/kg i.p. twice a day (n=6) for 21 days. RESULTS: Significant erythrocyte oxidant damage at baseline was indicated by: (i) dehydration, reduced cell K content, and up-regulated K-Cl co-transport; (ii) marked membrane externalization of phosphatidylserine; (iii) reduced plasma and membrane content of vitamin E; and (iv) increased membrane bound IgG. MonoHER treatment increased erythrocyte K content, and markedly improved all cellular indicators of oxidant stress and of lipid membrane peroxidation. While anemia did not improve, monoHER therapy reduced reticulocyte counts, improved survival of a fraction of red cells, and reduced ineffective erythropoiesis with decreased total bilirubin, lactate dehydrogenase and plasma iron. INTERPRETATION AND CONCLUSIONS: Antioxidant therapy reverses several indicators of oxidant damage in vivo. These promising antioxidant effects of monoHER should be investigated further.

Mechanisms of band 3 oxidation and clustering in the phagocytosis of Plasmodium falciparum-infected erythrocytes.

Erythrocytes (RBCs) opsonized by IgG and complement are prevalently recognized and phagocytosed by complement receptor CR1. This mechanism, effective in senescent and damaged RBCs seems to be operative in ring-parasitized RBCs, since infection by Plasmodium falciparum induces stage-dependent binding of auto-antibodies and activated C3 to the RBC membrane. Later, parasite forms are also recognized by non-opsonic receptors, such as scavenger receptor CD36. Malaria parasites induce the oxidative formation of hemichromes which are the trigger for the auto-antigen development. Band 3 protein is the most plausible candidate of the RBC auto-antigen, induced by hemichromes. Auto-antigens isolated from trophozoites were found only in a high-molecular-weight protein aggregates not present in the normal RBC. The immunocomplex was purified by protein-A affinity chromatography, purified proteins digested by trypsin and analyzed by MALDI-TOF. Peptide mapping showed that the main antigen consisted of band 3 protein aggregates that also contained hemichromes, IgGs, complement factor 3 (C3), and traces of spectrin and glycophorin but no parasite proteins. Two cysteines located in the band 3 cytoplasmic domain were found to be particularly reactive to oxidants and mediated band 3 covalent dimerization via disulfide bonds. Thus, parasites promote oxidative alterations in the membrane of the host which lead to exposure of antigenic sites recognized by anti-band 3 auto-antibodies. Formation of band 3 clusters appears to be mediated by cytoplasmic binding of hemichromes and also by direct band 3 oxidation, whereby clustered, oxidized and antigenic band 3 was underglycosylated.

Characterization of sodium dodecylsulfate polyacrylamide gel electrophoresis-separated M. agalactiae membrane antigens by mass spectrometry.

Mycoplasma membrane proteins are generally designated according to their apparent molecular weight measured by SDS-PAGE. Several results about mycoplasma membrane antigens are conflicting because some doubts are emerging about the accuracy of the method utilised to identify the antigens. Aim of this work, was to characterise proteins separated after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)-mass spectrometry to allow an uncontroversial designation of the antigens. Fifteen proteins with molecular weights ranging from 15,000 to 80,000 Da had been excised from gel and their whole molecular weight and proteolytic pattern had been determined using MALDI-TOF. The peptide pattern obtained using trypsin digestion allowed us to identify LipA, P48, P59, P80 and P40. Some other proteins showed analogies to proteins of Mycoplasma genitalium or Mycoplasma pneumoniae the only Mycoplasmas completely sequenced. There wasn't a close correspondence between the SDS-PAGE apparent molecular weight (generally used to name the proteins), the gene derived calculated mass and the molecular weight of whole proteins measured by MALDI-TOF. Only micro sequence data obtained by MS/MS allowed us to identify LipC, described as one of the most important Mycoplasma agalactiae antigens. This protein was found in correspondence with the 50 kDa region, instead of the 25 kDa region, confirming a phenomenon that we previously described.