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N-Linked Glycoprotein Profiles of Human Breast Basal Malignant Cell Lines
Secreted proteins and transmembrane proteins with extracellular domains are frequently glycosylated; this group of proteins includes those that participate in the various intercellular junctions and signaling pathways of an epithelium. In this study we characterized the differences in glycoprotein expression between claudin-low and other breast cell lines using a dataset of 26 breast cell lines in which the glycoproteins were identified and quantitated by liquid chromatography/ tandem mass spectrometry. Our goals are to characterize the glycoproteome of a set of claudin-low lines, compare them to basal, luminal and non-malignant cells and to identify drugs that may be especially effective on these cell lines. These nine basal malignant breast cells (HCC1143, HCC1395, HCC1937, HCC1954, HCC38, HCC70, MDAMB468, SUM149PT and SUM229PE) data are a part of 26 breast cell lines we analyzed.
Sample Processing Protocol
Breast cells were grown in 10 cm culture dishes with 10ml of culture medium. Cells were grown at 37C with 5% CO2. Intact cells were treated with periodate to oxidize monosaccharides within the carbohydrate chains of secreted and cell surface glycoproteins. Normal, benign and breast cancer cell lines were grown to >95% confluence. The growth medium was aspirated from each plate of cells and the cells were rinsed once with phosphate buffered saline (PBS) buffer. Cells were oxidized with 10mM NaIO4 in coupling buffer (20mM sodium acetate, 150mM NaCl, pH 5) in the dark at 25C for 1 hour with gentle rocking and the periodate solution removed by aspiration. The cells were washed with PBS with gently rocking for 2 min and the PBS buffer removed. Periodate treated cells were incubated with 0.5ml of coupling buffer containing 1% octyl--D-1-thioglucopyranoside and 1% protease inhibitor cocktail, at room temperature for 1 h with gentle rocking. Cell residue was scraped from the dishes and homogenized by multiple passes through a syringe with a series of different needle sizes ranging from 19 to 27½ gauge. The lysates were centrifugation at 14,000 rpm for 8 minutes at 4C. The supernatant was collected, and frozen at -20C until the samples were enriched for glycoproteins using hydrazide magnetic beads. Breast cell lysates were spiked with 5µg of periodate oxidized chicken ovalbumin. Hydrazide magnetic beads were mixed with the lysate supernatant and shaken for 16 hours at 25ºC with a thermo mixer at 800 rpm. The magnetic beads were repeatedly washed with a series of three washing buffers (1.5M NaCl, 60% methanol, 60% acetonitrile). Proteins bound to the magnetic beads were denatured with 8M urea at 25ºC with constant mixing. A rinse of 50mM ammonium bicarbonate was used to remove the urea. The bound proteins were reduced with 50mM dithiothreitol for 40 minutes at 40C. After reduction, the beads were rinsed with 50mM ammonium bicarbonate buffer and the glycoproteins were alkylated with 1ml of 50mM iodoacetamide for 30 minutes at 25ºC in the dark with constant mixing. The iodoacetamide was discarded, and the immobilized glycoproteins rinsed with 1ml of ammonium bicarbonate buffer. The glycoproteins were digested with trypsin at 37ºC for 12h. After digestion, the tryptic fraction was collected, and processed by solid phase extraction (SPE). N-Glycopeptide Release from the Hydrazide Resin with N-Glycosidase F-The N-linked glycopeptides bound to the hydrazide magnetic beads were released from the beads with N-glycosidase F by incubating the solution overnight at 37C with constant mixing. The released N-linked glycopeptide fraction was collected and subsequently processed by SPE. Solid Phase Extraction-The tryptic peptide and N-linked glycopeptide fractions were each subjected to SPE (Strata-X reversed phase, Phenomenex). The SPE resin was activated with methanol and rinsed with water. The tryptic or PNGase F treated samples were loaded onto and bound to an SPE column. The resin-bound peptides were rinsed with water to remove salts, eluted with 65% methanol/water, dried using a Speed-Vac apparatus, and stored at 4C prior to LC-MS/MS analysis. Protein Identifications by LC-MS/MS Analysis- Tryptic peptides and the deglycosylated N-linked peptides derived from each cell lysate were separately analyzed using an extensive set of LC-MS/MS analyses using a LTQ or a QExactive MS to maximize glycoprotein identification and to improve the reproducible detection of glycoproteins. LTQ MS analysis- Samples were analyzed using a data-dependent scan procedure (triple play-top 4 CID, CID=35%) at MS scan range of m/z 400-1800 with 4 dynamic exclusion (DE) time of 30, 45, 60 and 90s to acquire MS/MS spectra. In addition, 3 gas fractionations of m/z 400-900, m/z 700-1200, or m/z 1000-1800 with DE=1 of 60s were also employed. A C18 column (75μm x 130mm) was used for LC/MS. The mobile phase A was 0.1% HCOOH/water and the mobile phase B was 0.1% HCOOH in acetonitrile. A gradient was used for separation (5% to 35% B in 65 min, 35% to 80% B in 10 min, holding at 80% B for 5 min, and back to hold at 5% B). On-line 2D-LC-MS/MS analyses were also conducted to separate the tryptic peptides into 6 fractions (20, 40, 60, 80, 200, 400mM) of NH4Cl. Each fraction was chromatically resolved by a 120min LC program. Q Exactive MS analysis-The same nanoLC setup for LTQ was used with the data dependent acquisition of DE = 10 s and MS/MS (HCD= 27eV) for top 10 abundant ions. Resolving power for Q Exactive was set as 70,000 for the MS scan, and 17,500 for the MS/MS scan at m/z 200. Four LC/MS/MS analysis were collected for the tryptic digest samples and the PNGase F treated samples.
Data Processing Protocol
The Mascot (v2.3) algorithm was used to identify peptides from the resulting MS/MS spectra by searching against the combined human protein database (a total of 22,673 proteins) extracted from SwissProt (v57.14; 2010 February) using taxonomy “homo sapiens”(22,670 proteins). In addition BSA and fetuin were include to provide a means for estimating the level of protein contamination resulting from fetal bovine serum proteins contained in the cell culture medium. Ovalbumin was included to estimate glycoprotein recovery. Searching parameters for parent-/fragment-ion mass tolerances were set as 1.2-1.6/0.6-0.8 Da for the LTQ MS, and 20 ppm/0.1 Da for the Q Exactive MS. Other parameters used were a fixed modification of carbamidomethyl-Cys, variable modifications of deamidation-Asn (or/and Gln), and oxidation-Met. Trypsin was set as the protease with amaximum of 2 missed cleavages. Scaffold (Proteome Software) was used to merge and summarize the data obtained from the LC/MS/MS protein identification analyses for the LTQ MS and the Q Exactive MS. Protein identifications were based on a minimum detection of 2 peptides with 99% protein identification probability using the algorithm ProteinProphet. Each peptide identified had a minimum peptide identification probability of 95% using the algorithm PeptideProphet. The false positive rate for the peptide identification in this study was less than 5% for LTQ and less than 1% for Q Exactive based on results obtained with the decoy database searching. ProteinID Finder (Proteome Solutions) was used to determine whether the peptide was derived from a glycoprotein from the UniProt database for each identified protein.
Yen TY, Bowen S, Yen R, Piryatinska A, Macher BA, Timpe LC. Glycoproteins in Claudin-Low Breast Cancer Cell Lines Have a Unique Expression Profile. J Proteome Res. 2017 Mar 21 PubMed: 28287265
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