Time course proteomic profiling of human myocardial infarction plasma samples: an approach to new biomarker discovery.

BACKGROUND: The aim of this study was to identify novel candidate biomarker proteins differentially expressed in the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a high throughput screening technology. METHODS: Ten individuals with recent acute ischemic-type chest pain (<12 h duration) and ST-segment elevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at six times after STEMI diagnosis. The first stage (T0) was in Emergency Unit before receiving any medication, the second was just after primary angioplasty (T2), and the next four stages occurred at 12 h intervals after T0. Individuals (n=7) with similar risk factors for cardiovascular disease and normal ergometric test were selected as a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intact proteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent). RESULTS: Compared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in the first 48 h after the AMI (p<0.05). The 2STEMI group, had ~85% fewer differently expressed protein peaks than those without previous history of AMI (76, p<0.05). Among the 16 differentially-regulated protein peaks common to both STEMI cohorts (compared with the CG at T0), 6 peaks were persistently down-regulated at more than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB) during 48 h-period after IAM. CONCLUSIONS: Proteomic analysis by SELDI-TOF-MS technology combined with bioinformatics tools demonstrated differential expression during a 48 h time course suggests a potential role of some of these proteins as biomarkers for the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery.

Time course proteomic profiling of human myocardial infarction plasma samples: An approach to new biomarker discovery

Background: The aim of this study was to identify novel candidate biomarker proteins differentially expressedin the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a highthroughput screening technology.Methods: Ten individuals with recent acute ischemic-type chest pain (b12 h duration) and ST-segmentelevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at sixtimes after STEMI diagnosis. The first stage (T0) was in Emergency Unit before receiving any medication, thesecond was just after primary angioplasty (T2), and the next four stages occurred at 12 h intervals after T0.Individuals (n=7) with similar risk factors for cardiovascular disease and normal ergometric test were selectedas a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intactproteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent).Results: Compared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in thefirst 48 h after the AMI (pb0.05). The 2STEMI group, had ~85% fewer differently expressed protein peaks thanthose without previous history of AMI (76, pb0.05). Among the 16 differentially-regulated protein peakscommon to both STEMI cohorts (compared with the CG at T0), 6 peaks were persistently down-regulated atmore than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB)during 48 h-period after IAM.Conclusions: Proteomic analysis by SELDI-TOF-MS technology combinedwith bioinformatics tools demonstrateddifferential expression during a 48 h timecourse suggests a potential role of someof these proteins as biomarkersfor the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery.

A molecular mimic of phosphorylated prolactin (S179D PRL) secreted by eukaryotic cells has a conformation with an increased positive surface charge compared to that of unmodified prolactin.

S179D prolactin (S179D PRL) is a pseudophosphorylated form of human PRL which has potent antitumor and anti-angiogenic activities in vivo. This molecule binds to the same forms of the PRL receptor (PRLR) as unmodified PRL, yet this binding results in different intracellular signaling and biological end points. Since it is now clear that PRLRs are predimerized and therefore that ligand binding must initiate signaling by inducing a conformational change in the receptor dimer, we hypothesized that S179D PRL had an altered conformation compared to unmodified PRL. The conformation of the ligand-receptor ternary complex would therefore also have an altered conformation, and thus, different signaling molecules would be activated. Here we present evidence in support of this hypothesis by demonstrating, in contrast to unmodified PRL, that S179D PRL has reduced nickel and zinc binding capacity and a higher affinity for heparin and DEAE. Conformational changes have occurred since these features are counterintuitive on the basis of the simple substitution of a serine with a negatively charged aspartate residue. To demonstrate that these particular properties of S179D PRL were not due to misfolding of the molecule during production, S179D PRL was expressed in two different mammalian cell lines. Also investigated was the potential for production of S179D PRL as a soluble cytoplasmic, or secreted periplasmic, protein in Escherichia coli.

Periplasmic expression of human growth hormone via plasmid vectors containing the lambdaPL promoter: use of HPLC for product quantification.

The influence of different factors acting on Escherichia coli periplasmic expression of recombinant human growth hormone (hGH) in shake flask cultures has been investigated. Bacterial vectors containing the phage lambdaP(L) promoter, which is temperature activated, were utilized. Four different signal peptides were compared: DsbA, npr, STII and one derived from the natural hGH signal peptide, this last used as a reference. Other factors such as medium composition, optimized induction and expression conditions, and different bacterial strains were also studied. The determination of hGH, carried out directly in osmotic shock fluids, was based on an isocratic reversed-phase high-performance liquid chromatography method, which allows direct, rapid evaluation of the quality and quantity of hGH being secreted in the bacterial periplasmic space immediately after or even during fermentation. The level of hGH production increased 2.5-fold compared with the reference vector, reaching a level of 3.9 +/- 0.63 micro g/ml/A(600) (n = 6; coefficient of variation = 16.2%). The expression level was affected by the signal peptide and by the induction conditions, being more effective when activation started in the early logarithmic phase which, however, exhibited remarkably different optical density (OD) according to medium composition. Our results thus indicate that 6 h activation at 40-42 degrees C, starting with an OD (A(600)) of approximately 3 in a very rich medium, were conditions capable of providing the maximum secretion level for a vector utilizing the DsbA signal sequence and E.coli W3110 or RB791 as host cells.