Acetylation of lysines on affinity-purification matrices to reduce co-digestion of bead-bound ligands v1

In mass-spectrometry-based interaction proteomics on-bead digestion protocols are commonly applied after affinity-enrichment due to their simplicity and high efficiency. However, on-bead digestion often leads to strong background signals due to co-digestion of the bead-bound ligands such as streptavidin or antibodies. We present an effective, rapid and low-cost method to specifically reduce the peptide signals from co-digested matrix ligands. A short pre-incubation of matrix beads with Sulfo-NHS-Acetate (S-NHS-Ac) leads to acetylation of free amines on lysine side-chains of the bead-bound ligands making them resistant to Lys-C-mediated proteolysis. After binding of bait proteins to the acetylated beads we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion with trypsin. The strong reduction of interfering ligand peptides improves signal strength and data quality for the peptides of interest in liquid chromatography mass spectrometry (LC-MS).

A Data-Dependent Acquisition Ladder for Ultrasensitive (Neuro)Proteomics

Measurement of broad types of proteins from a small number of cells to single cells would help to better understand the nervous system but requires significant leaps in high-resolution mass spectrometry (HRMS) sensitivity. Microanalytical capillary electrophoresis electrospray ionization (microCE-ESI) offers a path to ultrasensitive proteomics by integrating scalability with sensitivity. We report here a data acquisition strategy that expands the detectable and quantifiable proteome in trace amounts of digests using microCE-ESI-HRMS. Data-dependent acquisition (DDA) was programmed to progressively exclude high-intensity peptide signals during repeated measurements. These nested experiments formed rungs of our DDA ladder. The method was tested for replicates analyzing ~500 pg of protein digest from cultured hippocampal (primary) neurons (mouse), which estimates to the total amount of protein from a single neuron. Analysis of net amounts approximating to ~10 neurons identified 428 nonredundant proteins (415 quantified), an ~35% increase over traditional DDA. The identified proteins were enriched in neuronal marker genes and molecular pathways of neurobiological importance. The DDA ladder deepens the detectable proteome from trace amounts of proteins, expanding the analytical toolbox of neuroscience.

Enhancement of Tryptic Peptide Signals from Tissue Sections using MALDI IMS Post-ionization (MALDI-2)

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve sensitivity and depth of coverage for protein and peptide IMS. Laser-based post-ionization MALDI-2 has been shown to increase sensitivity for several molecular classes but thus far this has not been reported for peptides. Here, we demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (termed MALDI-1), and conventional MALDI augmented using a second ionizing laser (termed MALDI-2). Proteins were digested <i>in situ</i> using trypsin prior to IMS analysis. For identification of peptides and proteins, a tissue homogenate was analyzed by LC-MS/MS for bottom-up proteomics and the corresponding proteins identified. These proteins were next fully ‘digested <i>in silico’</i> to generate a database of theoretical peptides to then match to MALDI IMS datasets. Peptides were tentatively identified by matching the MALDI peak list to the database peptide list employing a 5 ppm error window. This resulted in 314 ± 45 (n=3) peptides and 1 112 ± 84 (n=3) peptides for MALDI-1 and MALDI-2, respectively. Protein identifications were similarly made by linking IMS data to the LC-MS/MS peptide database. With positive protein identifications requiring two or more peptides per protein, 55 ± 13 proteins were identified with MALDI-1 and 205 ± 10 with MALDI-2. These results demonstrate that MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and significantly increases the number of proteins identified in IMS experiments.<br>

Enhancement of Tryptic Peptide Signals from Tissue Sections using MALDI IMS Post-ionization (MALDI-2)

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve sensitivity and depth of coverage for protein and peptide IMS. Laser-based post-ionization MALDI-2 has been shown to increase sensitivity for several molecular classes but thus far this has not been reported for peptides. Here, we demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (termed MALDI-1), and conventional MALDI augmented using a second ionizing laser (termed MALDI-2). Proteins were digested <i>in situ</i> using trypsin prior to IMS analysis. For identification of peptides and proteins, a tissue homogenate was analyzed by LC-MS/MS for bottom-up proteomics and the corresponding proteins identified. These proteins were next fully ‘digested <i>in silico’</i> to generate a database of theoretical peptides to then match to MALDI IMS datasets. Peptides were tentatively identified by matching the MALDI peak list to the database peptide list employing a 5 ppm error window. This resulted in 314 ± 45 (n=3) peptides and 1 112 ± 84 (n=3) peptides for MALDI-1 and MALDI-2, respectively. Protein identifications were similarly made by linking IMS data to the LC-MS/MS peptide database. With positive protein identifications requiring two or more peptides per protein, 55 ± 13 proteins were identified with MALDI-1 and 205 ± 10 with MALDI-2. These results demonstrate that MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and significantly increases the number of proteins identified in IMS experiments.<br>

Enhancement of Tryptic Peptide Signals from Tissue Sections using MALDI IMS Post-ionization (MALDI-2)

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve sensitivity and depth of coverage for protein and peptide IMS. Laser-based post-ionization MALDI-2 has been shown to increase sensitivity for several molecular classes but thus far this has not been reported for peptides. Here, we demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (termed MALDI-1), and conventional MALDI augmented using a second ionizing laser (termed MALDI-2). Proteins were digested <i>in situ</i> using trypsin prior to IMS analysis. For identification of peptides and proteins, a tissue homogenate was analyzed by LC-MS/MS for bottom-up proteomics and the corresponding proteins identified. These proteins were next fully ‘digested <i>in silico’</i> to generate a database of theoretical peptides to then match to MALDI IMS datasets. Peptides were tentatively identified by matching the MALDI peak list to the database peptide list employing a 5 ppm error window. This resulted in 314 ± 45 (n=3) peptides and 1 112 ± 84 (n=3) peptides for MALDI-1 and MALDI-2, respectively. Protein identifications were similarly made by linking IMS data to the LC-MS/MS peptide database. With positive protein identifications requiring two or more peptides per protein, 55 ± 13 proteins were identified with MALDI-1 and 205 ± 10 with MALDI-2. These results demonstrate that MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and significantly increases the number of proteins identified in IMS experiments.<br>

Tau peptide signals are seen in Parkinson subjects by Broderick Probe® sensors

This “short but sweet” clinical paper is about a next generation “uptick” nanotechnology that demonstrates a unique, real time imaging inventive art enabling a different kind of look at the brain, actually, to see inside neuronal and glial circuitry in the brain and spinal cord of the living human being and animal. “Lewy body dementia is characterized by the abnormal buildup of proteins into masses known as Lewy bodies. This protein is also associated with Parkinson’s disease. People who have Lewy bodies in their brains also have the plaques and tangles associated with Alzheimer's disease. Apr 26, 2019, Mayo clinic”.1 This is the problem and thus far, the problem is addressed primarily after autopsy, called post mortem, also problematic. Therefore, the purpose of this paper is to introduce the online and real time and spatial sensitive voltaic image of the Tau peptide complex video-tracked by the BRODERICK PROBE® biomedical sensors in striatum of the living Parkinson subject. This nanoprobe enables studies of the striking consequences among intensities of phosphorylated Tau. Identifying Tau on line is unmistakably relevant to longevity, both individual and societal and this relevance is inexorably critical to and on behalf of humanity per se.

The role of CLV1, CLV2 and HPAT homologs in nitrogen-regulation of root development

Plants use a variety of hormonal and peptide signals to control root development, including in adapting root development to cope with nutrient stress. Nitrogen (N) is a major limiting factor in plant growth and in response to N stress plants dramatically modulate root development, including in legumes influencing the formation of N-fixing nodules in response to external N supply. Recently, specific CLE peptides and/or receptors important for their perception, including CLV1 and CLV2, have been found to play important roles in root development in a limited number of species, including in some cases the response to N. In the legume Medicago truncatula, this response also appears to be influenced by RDN1, a member of the hydroxyproline O-arabinosyltransferase (HPAT) family which can modify specific CLE peptides. However, it not known if this signalling pathway plays a central role in root development across species, in particular root responses to N. In this study, we sought to systematically examine the role of homologues of these genes in root development of the legume pea (Pisum sativum. L) and non-legume tomato (Solanum lycopersicum) using a mutant based approach. This included a detailed examination of root development in response to N in these mutant series in tomato. We found no evidence for a role of these genes in pea seedling root development. Furthermore, the CLV1-like FAB gene did not influence tomato root development, including N response. In contrast, both CLV2 and the HPAT FIN appear to positively influence root size in tomato but do not mediate root responses to N. These suggest a relatively species-specific role for these genes in root development, including N regulation of root architecture.

The Role of Peptide Signals Hidden in the Structure of Functional Proteins in Plant Immune Responses

Plants have evolved a sophisticated innate immune system to cope with a diverse range of phytopathogens and insect herbivores. Plasma-membrane-localized pattern recognition receptors (PRRs), such as receptor-like kinases (RLK), recognize special signals, pathogen- or damage-associated molecular patterns (PAMPs or DAMPs), and trigger immune responses. A growing body of evidence shows that many peptides hidden in both plant and pathogen functional protein sequences belong to the group of such immune signals. However, the origin, evolution, and release mechanisms of peptide sequences from functional and nonfunctional protein precursors, known as cryptic peptides, are largely unknown. Various special proteases, such as metacaspase or subtilisin-like proteases, are involved in the release of such peptides upon activation during defense responses. In this review, we discuss the roles of cryptic peptide sequences hidden in the structure of functional proteins in plant defense and plant-pathogen interactions.