MALDI mass spectrometry imaging is able to simultaneously determine the spatial distribution of hundreds of molecules directly from tissue sections, without labeling and without prior knowledge. Ultra-high mass resolution measurements based on Fourier-transform mass spectrometry have been utilized to resolve isobaric lipids, metabolites and tryptic peptides. Here we demonstrate the potential of 15T MALDI-FTICR MSI for molecular pathology in a mouse model of high-grade glioma. The high mass accuracy and resolving power of high field FTICR MSI enabled tumor specific proteoforms, and tumor-specific proteins with overlapping and isobaric isotopic distributions to be clearly resolved. The protein ions detected by MALDI MSI were assigned to proteins identified by region-specific microproteomics (0.8 mm2 regions isolated using laser capture microdissection) on the basis of exact mass and isotopic distribution. These label free quantitative experiments also confirmed the protein expression changes observed by MALDI MSI and revealed changes in key metabolic proteins, which were supported by in-situ metabolite MALDI MSI.

Mass spectrometry imaging (MSI) is able to simultaneously record the distributions of hundreds of molecules directly from tissue. Rapid direct tissue analysis is essential for MSI in order to maintain spatial localization and acceptable measurement times. The absence of an explicit analyte separation/purification step means MSI lacks the depth of coverage of LC-MS/MS. In this work, we demonstrate how atmospheric pressure MALDI-MSI enables the same tissue section to be first analyzed by MSI, to identify regions of interest that exhibit distinct molecular signatures, followed by localized proteomics analysis using laser capture microdissection isolation and LC-MS/MS.

Here we assessed the ability of an automated sample preparation device equipped with disposable microcolumns to prepare mass-limited samples for high-sensitivity quantitative proteomics, using both label-free and isobaric labeling approaches. First, we compared peptide label-free quantification reproducibility for 1.5-150 μg of cell lysates and found that labware preconditioning was essential for reproducible quantification of <7.5 μg digest. Second, in-solution and on-column tandem mass tag (TMT) labeling protocols were compared and optimized for 1 μg of sample. Surprisingly, standard methods for in-solution and on-column labeling showed poor TMT labeling (50-85%); however, novel optimized and automated protocols restored efficient labeling to >98%. Third, compared with a single long gradient experiment, a simple robotized high-pH fractionation protocol using only 6 μg of starting material doubled the number of unique peptides and increased proteome coverage 1.43-fold. To facilitate the analysis of heterogeneous tissue samples, such as those obtained from laser capture microdissection, a modified BCA protein assay was developed that consumes and detects down to 15 ng of protein. As a proof-of-principle, the modular automated workflow was applied to 0.5 and 1 mm mouse kidney cortex and medulla microdissections to show the method's potential for real-life small sample sources and to create kidney substructure-specific proteomes.

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We studied the interactions of rabbit glue, a collagen-based proteinaceous binder, with azurite (Cu3(CO3)2(OH)2), calcium carbonate (CaCO3), hematite (Fe2O3·nH2O), red lead (Pb3O4) and cinnabar (HgS) by Fourier transform infrared spectroscopy (FT-IR). The research was carried out on a set of paint reconstructions, which were analysed before and after artificial light ageing. A deconvolution of the amide I FT-IR absorption peak was performed with a written-in-house LabVIEW program to study the secondary structure of the glue.The changes in the glue conformation highlighted that all the inorganic pigments interact with the proteinaceous binder. The conformational changes were correlated with a loss of stability of the collagen structure, especially after ageing, likely due to the interlayer coordination of metals salts and oxide with protein functional groups. These results were correlated with the lower thermal stability of the glue/pigment mixtures with respect to the pure glue, evidenced by thermogravimetry (TG) and differential scanning calorimetry (DSC) analyses performed in a previous step of this work

are linked to a baleful prognosis, with a survival average of 6-10 mo after surgery [3,4] . Many studies have demonstrated that only surgery can lead to a control of chronic anemia related to intestinal melanoma bleeding and resolution of the episodes of intestinal sub-occlusion. Surgery on melanoma metastases moreover, can guarantee an increase of sur vival, in addition to an excellent improvement in quality of life [5][6][7] .Intestinal metastases represent the occurrence of an occult skin melanoma in only 3%-5% of cases, in which a spontaneous regression of the cutaneous lesion happens [8] .Intestinal metastasis bleeding is extremely rare [9,10] . In order to add more information about surgical presentation of intestinal occult melanoma herein we describe a case of a young woman affected by bloody jejunal metastasis of occult cutaneous melanoma, complicated by intestinal invagination-an extremely rare case in the adult population. CASE REPORTA 45-year old woman complained of continual nausea and biliary vomiting, associated with a weight loss of 5 kg. Due to localized abdominal pain, mainly in the right hypochondrium and episodes of hematemesis, the patient was admitted to our hospital. Blood tests revealed sideropenic anemia with 81 g/L haemoglobin, serum iron 100 pg/L, ferritin 23 µg/L, and fecal occult blood test (FOBT) positive in three fecal samples. A gastroscopy was performed, which showed the presence of gradeⅠesophagitis, moderate hiatal hernia and chronic erosive gastritis. The colonoscopy was incomplete due to the presence of colic stools. Abdominal ultrasonography highlighted a distension of the intestinal loops without signs of parenchymatous organ pathology. The patient therefore received an abdominal CT, which suggested the presence of a gastric distension with duodenum-jejunal distension and the presence of a jejunal loop with thickened walls. A second hyperdense image inside the intestinal lumen, forming a targetshaped image was also present: typical feature of intestinal invagination (Figure 1). We therefore decided to proceed to urgent surgical operation after blood transfusion. During surgery, intra-peritoneal fluid was found and samples were removed for cytological testing. Invagination at the third jejunal loop (Figure 2) AbstractCutaneous melanoma is one of the most studied neoplastic lesions in biology and clinical oncology. It has been well documented that this type of neoplasm presents a high metastatic rate, and is able to involve nearly every tissue. Non-cutaneous melanoma represents an unusual pattern of melanoma, and the small intestine is an uncommon anatomic localization. Herein we report an extremely rare clinical case of a young woman affected by a bleeding jejunal melanoma, whose early clinical presentation was an intestinal invagination.

Atmospheric pressure MALDI on a Q-Exactive instrument was optimized for in-source decay and pseudo-MS3. The dependence of AP-MALDI ISD on the MALDI liquid matrix was investigated for peptides and proteins. The liquid matrices enabled long-life ISD signal, and exhibited high fragment ion yield and signal stability. Extensive a-, b-, c-, y-, and z-type fragment series were observed depending on the matrix used but were most extensive with 2,5-DHB. Complete sequence coverage of small peptide and intact protein-terminus sequence tags were obtained and confirmed using HCD as a pseudo-MS3 method. Graphical Abstractᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1511-0) contains supplementary material, which is available to authorized users.

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