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Constitutive Androstane Receptor

While several MS-based studies of MM have been undertaken previously,16C19 our study offers several advantages, including the use of CSC to specifically detect proteins present within the cell surface, the use of multiple cell lines to account for possible differences among individuals, and the use of both PRM and FCM to determine if proteins of interest discovered on immortalized cell lines are relevant to primary human MM cells

While several MS-based studies of MM have been undertaken previously,16C19 our study offers several advantages, including the use of CSC to specifically detect proteins present within the cell surface, the use of multiple cell lines to account for possible differences among individuals, and the use of both PRM and FCM to determine if proteins of interest discovered on immortalized cell lines are relevant to primary human MM cells. PRM analyses recognized 30 proteins significantly higher in abundance in the CD138+ cells from MM patients compared with their CD138? subsets. non-specifically bound peptides. By digestion with PNGaseF, the peptides were released from your glycan moiety and then consequently desalted and dried under vacuum. Samples were analyzed using a Q Exactive MS (Thermo; Waltham, Massachusetts, USA). Data were analyzed using ProteomeDiscoverer 2.2 (Thermo). The exported peptide lists were manually examined and proteins that lacked (±)-WS75624B at least one peptide having a deamidated asparagine within the N-linked glycosylation consensus sequence (N-X-S/T/C where X is definitely any amino acid except proline) were discarded (on-line supplementary table 1). Supplementary data jitc-2020-000915supp001.xlsx Cell lysis, protein digestion, and peptide clean-up For whole-cell lysate analysis of lymphocyte cell lines and patient samples, pellets of cells were lysed in 500?L of 2X Invitrosol (40%?v/v; Thermo Fisher Scientific) and 20% acetonitrile in 50?mM Rabbit Polyclonal to PDGFRb ammonium bicarbonate. Samples were sonicated (VialTweeter; Hielscher (±)-WS75624B Ultrasonics, Teltow, Germany) by three 10-second pulses, arranged on snow for 1?min, and then resonicated. Beads were removed magnetically. Samples were brought to 5?mM tris(2-carboxyethyl)phosphine (TCEP) and reduced for 30?min at 37C on a Thermomixer at 1200 RPM. Samples were then brought to 10?mM iodoacetamide (IAA) and alkylated for 30?min at 37C on a Thermomixer at 1200 RPM in the dark. 20 g of trypsin was added to each sample; digestion occurred over night at 37C on a Thermomixer at 1200 RPM. Peptides were (±)-WS75624B washed by SP2 following a standard protocol.23 Targeted quantitation of proteins of interest among cell lines and primary human being cells All targeted analyses were performed using an Orbitrap Fusion Lumos Tribrid MS (Thermo; for a full description observe online supplementary methods). Data were imported into Skyline24 and chromatographic peaks were extracted from MS2 spectral data for each recognized peptide from the prospective list. Statistical analyses were performed using College students t-test and plots were generated in GraphPad Prism. Supplementary data jitc-2020-000915supp002.pdf Results Cell surface N-glycoproteome of MM cell lines Four cell lines derived from MM individuals (RPMI-8226, RPMI-8226/R5, U-266, MM.1R) were analyzed. Two B cell lines (RPMI-1788, BLCL) were included for assessment. By applying CSC technology, 846 unique cell surface N-glycoproteins were recognized, including 171 cluster of differentiation (CD) antigens (on-line supplementary table 2). The list of 846 includes single-pass and multi-pass membrane proteins, glycophosphatidylinositol (GPI)-anchored proteins, and lipid-anchored proteins (number 1A). Overall, 81% of the proteins identified are known to be membrane-associated, demonstrating a high-quality enrichment for surface-localized proteins in the dataset. Open in a separate window Number 1 Overview of cell surface N-glycoproteins recognized by (±)-WS75624B cell surface capture analysis of multiple myeloma (MM) and B cell lines. (A) Distribution of protein types (±)-WS75624B recognized within each cell collection based on UniProt annotations for cluster of differentiation antigen notations and membrane, single-pass and multi-pass, glycophosphatidylinositol (GPI)-anchored and lipid-anchored proteins. (B) Upset storyline54 showing distribution of protein observations among B and MM cell lines. BLCL, B-lymphoblastoid cell collection. Supplementary data jitc-2020-000915supp003.xlsx Of 696 proteins identified within the 4?MM cell lines, 104 proteins were common to all lines. Many of these 104 proteins were also found on one or both B cell lines, with 7 proteins found specifically on all 4?MM cell lines (number 1B). This discovery-driven display recognized hematopoietic and B cell markers (eg, human being leukocyte antigen (HLA), IgM, CD80), and known MM markers, such as CD38, in addition to proteins not previously explained on MM cells. Further assisting the energy of our approach for identifying cell surface proteins with relevance to MM, we compared our results to a panel of known MM antigens. Seven proteins known to be helpful for immunophenotyping and monitoring of MM (BCMA, CD28, CD33, CD38, CD44, CD45, and CD54) were recognized by CSC, as expected. A further nine proteins (CD19, CD20, CD27, CD52, CD56, CD81, CD117, CD200, CD307) were not.