Additional file 1: Supplementary data on the synthesis of BODIPY-BBN, its in-vitro characterization and its in-vivo biodistribution and stability. Figure S1. of Development of a clickable bimodal fluorescent/PET probe for in vivo imaging

Synthesis and analytical data of BODIPY-azide 1. Figure S2. Synthesis of BODIPY-BBN 5 using CuAAC click chemistry. Figure S3. In vitro cell binding study of BODIPY-BBN 5 using GRPr overexpressing PC-3 cells. Figure S4. Quantification of tumor uptake comparing fluorescent imaging as well as gamma-counting. Figure S5. In vivo small animal PET/CT images (60 min) of PC-3 tumor-bearing nude mice after intravenous injection of 18F-BODIPY-BBN 3 (55 ± 10 μCi) in PBS (4 % DMSO, 200 μL). Figure S6. Percent intact fluorescent 19F-BODIPY-BBN in human serum at different time points (0, 15, 30, 45, and 60 min) after incubation at 37 °C

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

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

The differential serum denaturation protocol.

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

Effects of differential denaturation on serum electrophoretic separation.

<p>A: The serum protein pattern as described in literature and as obtained with DENT1 protocol in a 16×18 cm gradient SDS-PAGE (2.4–15%), shown in panel B, by MALDI-TOF/MS analysis of cut and digested protein bands. C: Representative electrophoretic separation of human pooled sera derived from the most discriminating sample pre-treatments (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057104#pone.0057104.s001" target="_blank">Table S1</a>, thereafter defined as DENT2 and DENT3 treatments) compared to the reference treatments (DENT1). Samples were run on a manually poured gradient slab gel (2,4–15%): 120 µg of proteins loaded per lane. Asterisks indicate some of the protein bands undetectable or less detectable in DENT1 but evident in the pre-treatments specifically developed in the present study. D: Graphical representation of the improvement of protein band resolution and discrimination after DENT2 or DENT3 pre-treatments compared to the DENT1. The protein pattern identified as Merge indicates the gain of protein band detection and discrimination.</p