An Alternative Paper Based Tissue Washing Method for Mass Spectrometry Imaging: Localized Washing and Fragile Tissue Analysis

Surface treatment of biological tissue sections improves detection of peptides and proteins for mass spectrometry imaging. However, liquid surface treatments can result in diffusion of surface analytes and fragile tissue sections can be easily damaged by typical washing solvents. Here, we present a new surface washing procedure for mass spectrometry imaging. This procedure uses solvent wetted fiber-free paper to enable local washing of tissue sections for mass spectrometry imaging and tissue profiling experiments. In addition, the method allows fragile tissues that cannot be treated by conventional washing techniques to be analyzed by mass spectrometry imaging

Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5 beta and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule- stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules

Challenging the heterogeneity of disease presentation in malignant melanoma-impact on patient treatment

There is an increasing global interest to support research areas that can assist in understanding disease and improving patient care. The National Cancer Institute (NIH) has identified precision medicine-based approaches as key research strategies to expedite advances in cancer research. The Cancer Moonshot program ( https://www.cancer.gov/research/key-initiatives/moonshot-cancer-initiative ) is the largest cancer program of all time, and has been launched to accelerate cancer research that aims to increase the availability of therapies to more patients and, ultimately, to eradicate cancer. Mass spectrometry-based proteomics has been extensively used to study the molecular mechanisms of cancer, to define molecular subtypes of tumors, to map cancer-associated protein interaction networks and post-translational modifications, and to aid in the development of new therapeutics and new diagnostic and prognostic tests. To establish the basis for our melanoma studies, we have established the Southern Sweden Malignant Melanoma Biobank. Tissues collected over many years have been accurately characterized with respect to the tumor and patient information. The extreme variability displayed in the protein profiles and the detection of missense mutations has confirmed the complexity and heterogeneity of the disease. It is envisaged that the combined analysis of clinical, histological, and proteomic data will provide patients with a more personalized medical treatment. With respect to disease presentation, targeted treatment and medical mass spectrometry analysis and imaging, this overview report will outline and summarize the current achievements and status within malignant melanoma. We present data generated by our cancer research center in Lund, Sweden, where we have built extensive capabilities in biobanking, proteogenomics, and patient treatments over an extensive time period

Strong cation exchange-based fractionation of Lys-N-generated peptides facilitates the targeted analysis of post-translational modifications

\u3cp\u3eIn proteomics multi-dimensional fractionation techniques are widely used to reduce the complexity of peptide mixtures subjected to mass spectrometric analysis. Here, we describe the sequential use of strong cation exchange and reversed phase liquid chromatography in the separation of peptides generated by a relatively little explored metallo-endopeptidase with Lys-N cleavage specificity. When such proteolytic peptides are subjected to low-pH strong cation exchange we obtain fractionation profiles in which peptides from different functional categories are well separated. The four categories we distinguish and are able to separate to near completion are (I) acetylated N-terminal peptides; (II) singly phosphorylated peptides containing a single basic (Lys) residue; (III) peptides containing a single basic (Lys) residue; and (IV) peptides containing more than one basic residue. Analyzing these peptides by LC-MS/MS using an ion trap with both collision as well as electron transfer-induced dissociation provides unique optimal targeted strategies for proteome analysis. The acetylated peptides in category I can be identified confidently by both CID and ETcaD, whereby the ETcaD spectra are dominated by sequence informative Z-ion series. For the phosphorylated peptides in category II and the normal single Lys containing peptides in category III ETcaD provides unique straightforward sequence ladders of c'-ions, from which the exact location of possible phosphorylation sites can be easily determined. The later fractions, category IV, require analysis by both ETcaD and CID, where it is shown that electron transfer dissociation performs relatively well for these multiple basic residues containing peptides, as is expected. We argue that the well resolved separation of functional categories of peptides observed is characteristic for Lys-N-generated peptides. Overall, the combination of Lys-N proteolysis, low-pH strong cation exchange, and reversed phase separation, with CID and ETD induced fragmentation, adds a new very powerful method to the toolbox of proteomic analyses.\u3c/p\u3