Targeting the hydrophilic regions of recombinant proteins by MS via in-solution buffer-free trypsin digestion.

A desalting step using reversed phase chromatography is a common practice prior to mass spectrometry analysis of proteolytic digests in spite of the detrimental exclusion of the hydrophilic peptides. The detection of such peptides is also important for the complete coverage of protein sequences and the analysis of posttranslational modifications as inquired by regulatory agencies for the commercialization of biotechnological products. The procedure described here, named in-solution buffer-free digestion, simplifies the sample processing and circumvents the above-mentioned limitations by allowing the detection of tryptic hydrophilic peptides via direct ESI-MS analysis. Two DNA recombinant proteins such as HBcAg (hepatitis B core antigen) and fusion VEGF (vascular endothelial growth factor) were analyzed with the proposed in-solution buffer-free digestion allowing the detection of extremely hydrophilic di-, tri- and tetra-peptides, C-terminal His-tail peptide, as well as disulfide-containing peptides. All these molecular species are hardly seen in mass spectrometric analysis using a standard digestion that includes a C18-desalting step. The procedure was also successfully tried on hydrophilic tetra- and hexa-peptides of Ribonuclease B carrying an N-glycosylation site occupied with "high-mannose" N-glycan chains. The in-solution buffer-free digestion constitutes a simple and straightforward approach to analyse the hydrophilic proteolytic peptides which are commonly elusive to the detection by conventional mass spectrometric analysis.

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.

SCX charge state selective separation of tryptic peptides combined with 2D-RP-HPLC allows for detailed proteome mapping.

Multidimensional peptide fractionation is widely used in proteomics to reduce the complexity of peptide mixtures prior to mass spectrometric analysis. Here, we describe the sequential use of strong cation exchange and reversed phase liquid chromatography in both basic and acidic pH buffers for separating tryptic peptides from complex mixtures of proteins. Strong cation exchange exclusively separates peptide by their charge state into neutral, singly and multi-charged species. To further reduce complexity, each peptide group was separated by reversed phase liquid chromatography at basic pH and the resultant fractions were analyzed by LC-MS/MS. This workflow was applied to a soluble protein lysate from mouse embryonic fibroblast cells, and more than 5000 proteins from 29,843 peptides were identified. The high selectivity displayed during the SCX step (93% to 100%) and the overlaps between proteins identified from the SCX-separated peptide groups, are interesting assets of the procedure. BIOLOGICAL SIGNIFICANCE: The present work shows how complex mixture of peptides can be selectively separated by SCX based essentially on the net charge of peptides. The proposed workflow results in three well-defined subset of peptides of specific amino acid composition, which are representative of the constituent proteins. The very high selectivity obtained (93% to 99%) on the peptide side, underscores for the first time the possibility of SCX chromatography to aid in validating identified peptides.

Isoelectric point optimization using peptide descriptors and support vector machines.

IPG (Immobilized pH Gradient) based separations are frequently used as the first step in shotgun proteomics methods; it yields an increase in both the dynamic range and resolution of peptide separation prior to the LC-MS analysis. Experimental isoelectric point (pI) values can improve peptide identifications in conjunction with MS/MS information. Thus, accurate estimation of the pI value based on the amino acid sequence becomes critical to perform these kinds of experiments. Nowadays, pI is commonly predicted using the charge-state model [1], and/or the cofactor algorithm [2]. However, none of these methods is capable of calculating the pI value for basic peptides accurately. In this manuscript, we present an new approach that can significant improve the pI estimation, by using Support Vector Machines (SVM) [3], an experimental amino acid descriptor taken from the AAIndex database [4] and the isoelectric point predicted by the charge-state model. Our results have shown a strong correlation (R(2)=0.98) between the predicted and observed values, with a standard deviation of 0.32 pH units across the complete pH range.

Charge state-selective separation of peptides by reversible modification of amino groups and strong cation-exchange chromatography: evaluation in proteomic studies using peptide-centric database searches.

Here we describe an integrated approach for the selective separation of peptides from complex mixtures using strong cation-exchange chromatography. The procedure exploits the charge differences produced by reversible modification of primary amino groups in peptides, enabling their separation into three major fractions: 1) neutral peptides 2) peptides with one positive charge and 3) peptides with 2 or more positive charges. The procedure demonstrated an excellent selectivity which allowed restricted MS/MS ion searches with peptide-centric databases.