Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDITOF/ MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: alpha 2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05). Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera

Intradermal lymphoscintigraphy at rest and after exercise: A new technique for the functional assessment of the lymphatic system in patients with lymphoedema

AIM: The aim of this study was to evaluate the effect of implementing a new technique, intradermal injection lymphoscintigraphy, at rest and after muscular exercise on the functional assessment of the lymphatic system in a group of patients with delayed or absent lymph drainage. Methods: We selected 44 patients (32 women and 12 men; 15 of 44 with upper limb and 29 of 44 with lower limb lymphoedema). Thirty of 44 patients had bilateral limb lymphoedema and 14 of 44 had unilateral disease; 14 contralateral normal limbs were used as controls. Twenty-three patients had secondary lymphoedema after lymphadenectomy and the remaining 21 had idiopathic lymphoedema. Each of the 44 patients was injected with 50MBq (0.3-0.4ml) of Tc-albumin-nanocolloid, which was administered intradermally at the first interdigital space of the affected limb. Two planar static scans were performed using a low-energy general-purpose collimator (acquisition matrix 128×128, anterior and posterior views for 5min), and in which drainage was slow or absent, patients were asked to walk or exercise for 2min. A postexercise scan was then performed to monitor and record the tracer pathway and the tracer appearance time (TAT) in the inguinal or axillary lymph nodes. Results: The postexercise scans showed that (i) 21 limbs (15 lower and six upper limbs) had accelerated tracer drainage and tracer uptake in the inguinal and/or axillary lymph nodes. Two-thirds of these showed lymph stagnation points; (ii) 27 limbs had collateral lymph drainage pathways; (iii) in 11 limbs, there was lymph drainage into the deeper lymphatic channels, with unusual uptake in the popliteal or antecubital lymph nodes; (iv) six limbs had dermal backflow; (v) three limbs did not show lymph drainage (TAT=not applicable). TAT=15±3min, ranging from 12 to 32min in limbs with lymphoedema versus 5±2min, ranging from 1 to 12min in the contralateral normal limbs (P&lt;0.001). Conclusion: Intradermal injection lymphoscintigraphy gives a better imaging of the lymph drainage pathways in a shorter time, including cases with advanced lymphoedema. In some patients with lymphoedema, a 2-min exercise can accelerate tracer drainage, showing several compensatory mechanisms of lymph drainage. The effect of the exercise technique on TAT and lymphoscintigraphy findings could result in a more accurate functional assessment of lymphoedema patients. © 2010 Wolters Kluwer Health | Lippincott Williams &amp; Wilkins

Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

<div><p>Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05).</p> <p>Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.</p> </div

MALDI-TOF/MS analysis of differentially expressed bands in melanoma patients.

<p>MALDI-TOF/MS analysis of differentially expressed bands in melanoma patients.</p><p>Differentially expressed bands by TRIDENT-SDS-PAGE identified by MALDI-TOF/MS in human sera from cancer patients compared to the healthy controls. P value means the significance between densitometry of control (Ctrl) bands <i>vs</i> melanoma bands (Mel), whose modulation is reported as State change. Prot ID describes the name of the protein and AC# the accession number.</p

Human serum protein bands resolution.

<p>Human pool serum bands resolution in the gradient vertical slab gel 2.4–15%, 16×18 cm, and effects of TRIDENT analysis on the gel bands discrimination. It is noteworthy that the total numbers of bands detectable under DENT2 (53±1) or DENT3 (59±2) pre-treatment reflect the improvement of bands resolution compared to the standard denaturation protocol DENT1 (36±1) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057104#pone-0057104-g002" target="_blank">Figure 2D</a>).</p

The differential serum denaturation protocol.

<p>Schematic representation of chemical-physical pre-treatments of sera and following analytical procedures.</p

Human serum protein bands resolution differentially denatured.

<p>Human serum protein bands resolution differentially denatured.</p><p>Estimation of serum protein bands resolution of serum differentially denatured with 3 different protocols, run onto 8×8 cm 2.5–15% gradient gels, visualized by silver staining protocol. Data are reported as mean ±SD.</p

Some serum proteins identified by TRIDENT-SDS PAGE compared to bibliographic references.

<p>Some of human and murine serum proteins identified with MALDI-TOF/MS are listed. For each identified protein, the following information is reported: PROT. (protein name), S (source, H = human, M = mouse), %C (percentage coverage), #P (number of unique peptides identified), ANN (annotations with theoretical MW and NCBI protein accession number), DENT (denaturation treatment used), mass errors for each sequence analysed, MSC (mass score), #MVS (number of mass values searched), #MVM (number of mass values matched), MW T and MW O (theoretical and observed molecular weights, respectively) and CONC (the serum/plasma concentration levels known by literature).</p

LC-MS/MS analysis of differentially expressed bands in melanoma patients.

<p>Some of human serum proteins whose expression was significantly different in melanoma <i>vs</i> control sera were further identified by LC-MS/MS. For each identified protein, the following information are reported: Reference = name as reported in annotations; P(pro) = peptide probability; Score; Coverage = percentage of coverage; MW = theoretical molecular weight; Accession = NCBI protein accession number; Peptide = number of unique peptides identified.</p

MALDI-TOF/MS analysis of differentially expressed bands in melanoma carrying mice.

<p>Differentially expressed bands by TRIDENT-SDS-PAGE identified by MALDI-TOF/MS in murine sera from cancer animal compared to the healthy controls. P value means the significance between densitometry of control (Ctrl) bands <i>vs</i> melanoma bands (Mel), whose modulation is reported as State change. Prot ID describes the name of the protein and AC# the accession number.</p