Identifying the CHO Secretome using Mucin-type O-Linked

Article pubs.acs.org/jpr

Identifying the CHO Secretome using Mucin-type O‑Linked Glycosylation and Click-chemistry Peter G. Slade,*,† Mahbod Hajivandi,‡ Cheryl Moody Bartel,† and Stephen F. Gorfien§ †

Life Technologies, Eugene, Oregon 97401, United States Life Technologies, Carlsbad, California 92008, United States § Life Technologies, Grand Island, New York 14072, United States ‡

S Supporting Information *

ABSTRACT: Chinese hamster ovary cells (CHO) are the most common cell line used in the production of therapeutic proteins. Understanding the complex pattern of secreted host cell proteins (HCP) that are released by CHO cells will facilitate the development of new recombinant protein production processes. In this study, we have adapted the N-azido-galactosamine (GalNAz) metabolic labeling method to enable the mass spectrometry identification and quantification of secreted proteins in cell culture media. CHO DG44 and CHO-S cells were cultured in media containing GalNAz, which was metabolically incorporated into mucin-type O-linked glycans of secreted proteins. These proteins were effectively enriched using click-chemistry from the cell culture media, allowing for the analysis of secreted proteins across multiple days of cell growth. When compared to the standard method for secretome analysis, the GalNAz method not only increased the total number of proteins identified but dramatically improved the quality of data by decreasing the number of background proteins (cytosolic or nuclear) to essentially zero. KEYWORDS: CHO, DG44, CHO-S, secretome, host cell proteins, GalNAz, click-chemistry, O-linked glycosylation

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the CHO-K1 cell line has only recently been published.10 Increasing these resources could potentially aid in understanding and improving recombinant protein production processes to help enhance therapeutic protein development.2,11 In particular, a CHO proteome has yet to be well established and the few CHO proteomic studies performed to-date have generally focused on changes in the CHO intracellular proteome relative to the expression level of the recombinant protein.1,3 An important area of the proteome typically overlooked even though it plays an integral role in cell culture media conditions is the cellular secretome, often called host cell proteins (HCP) in conditioned media of transfected cells. The secretome generally refers to the collection of proteins that contain the signal peptide and are processed through the classical secretory pathway. It can also include proteins shed from the cell surface as well as proteins released by nonclassical methods. The secretome includes numerous proteins that regulate cell-to-cell and cell-to-extracellular matrix interactions, which affect cell growth, differentiation and angiogenesis. In cell lines used for biotherapeutic production, secreted proteins or HCP are considered necessary for cell growth and therapeutic protein production, but also pose potential problems for recombinant protein stability and purification, and may have detrimental effects (such as immunogenicity) if not removed from the final drug product.12,13 To ensure product purity,

INTRODUCTION Chinese hamster ovary (CHO) cells are one of the most common host cell lines for the production of therapeutic proteins, accounting for more than 70% of all current therapeutics. 1−3 The wide use of CHO cells in the biotherapeutic industry began after the isolation of cell lines with gene mutations that exhibited nutritional requirements for maintaining growth and viability. In particular, mutations in the dihydrofolate reductase (DHFR) gene resulted in the CHO cell lines DXB11 and DG44.4,5 DHFR-deficient cells facilitated the expression of genes cotransfected with a functional copy of the DHFR gene and allowed for the use of a DHFR inhibitor to increase the gene copy number, and therefore the productivity, of cells.6 The DXB11 and DG44 cell lines are some of the most widely used CHO cell host systems for protein production.7 Another common CHO cell line used in the biotechnology industry originated from Los Alamos National Laboratory (LANL). This cell line, called CHO-S, was derived from the original Chinese Hamster ovary isolate,8 and is distinguished from the CHO-K1 cell line based on karyotype analysis.9 Life Technologies received CHO-S cells from LANL in 1991; it was originally an adherent cell line that was propagated in growth medium supplemented with fetal bovine serum. Life Technologies subsequently adapted CHO-S cells to grow in suspension and removed the need for animal-based growth media. Unfortunately, unlike other rodent cells, CHO cell genetic and proteomic data are limited and the genomic sequence for © 2012 American Chemical Society

Received: August 27, 2012 Published: November 10, 2012 6175

dx.doi.org/10.1021/pr300810f | J. Proteome Res. 2012, 11, 6175−6186

Journal of Proteome Research

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comprehensive analysis of the CHO cell secretome generated to-date.

most of the pharmaceutical industry uses enzyme-linked immunosorbance assay (ELISA) to measure HCP levels during recombinant protein drug recovery; more recently, solid phase proximity ligation assay (PLA) has been demonstrated to be useful for this purpose, exhibiting several advantages over ELISA.14 It is important to question if HCP can be appropriately monitored using the same immunoassay for multiple CHO cell lines.15 Furthermore, understanding which secreted proteins promote growth and cell viability can play a significant role in developing new defined growth media. For example, the typical process of clone selection involves dilution to low cell densities, which frequently requires the inclusion of conditioned media (media containing an undefined number of secreted proteins). Ideally, it would be best to supplement media with defined amounts of known proteins that help promote single cell growth in order to have a more defined and therefore better controlled process. Current methods used to study secreted proteins are limited by the abundance of nonsecreted proteins occurring from either serum-supplemented media or from autolysis due to normal cell death. In the standard method of secretome analysis, cells are cultured to a desired density, washed with PBS or serum-free media, and then incubated in new or fresh serum-free media for a few hours.16 The proteins secreted into this media are commonly analyzed and characterized by 2-D gel electrophoresis or multidimensional protein identification technology (MudPIT) proteomics methods. Although incubation of cells in new or fresh media likely reduces interference from nonsecreted proteins, the number and variety of secreted proteins identified is limited by the incubation time, essentially giving only a snapshot of protein secretion during a finite time point. Improving methods for the identification of secreted proteins, specifically over longer durations or at different stages of cell growth, would greatly facilitate the development of new defined media technologies for biotherapeutic cell lines. To improve our ability to identify and quantify CHO cells’ secreted proteins, this study took advantage of the metabolic incorporation of the N-acetyl-galactosamine sugar analog containing an azide functional group (GalNAz or N-azidogalactosamine), first introduced by Bertozzi et al.17,18 The predominant form of O-linked glycosylation is the mucin-type, characterized by an initial N-acetylgalactosamine (GalNAc) residue linked to the hydroxyl group of either Thr or Ser side chains. It is generally accepted that mucin-type O-glycosylation is largely restricted to the cell surface and secreted proteins.19,20 CHO cells grown in media containing GalNAz incorporated this azide-sugar analogue into mucin-type O-linked glycans. Proteins proceeding through the typical secretory pathway were enriched using Cu(I) catalyzed click-chemistry to an agarose resin containing an alkyne functional group. After enrichment, secreted proteins were identified using MudPIT mass spectrometry proteomics and quantified by iTRAQ isotopic labeling.21 Applying this technology we successfully characterized and identified key differences in the secretome of CHO-S and DG44 cell lines. This methodology demonstrated a considerable improvement over the standard secretome analysis techniques by both increasing the number of secreted proteins identified and decreasing the number of background proteins (cytosolic or nuclear) to essentially zero. More importantly, it allowed for the continual monitoring of the secretome throughout growth. This study documents the most

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EXPERIMENTAL SECTION

Materials

CD FortiCHO medium, CD DG44 medium, glutamine, AntiClumping Agent, CHO-S (cGMP banked) and CHO DG44 (cGMP banked) parental cell lines are products of Life Technologies (Gibco). Click-iT GalNAz metabolic glycoprotein labeling reagent, Click-iT protein enrichment kit, Tetramethylrhodamine Alkyne (TAMRA), NuPAGE Novex 4−12% Bis-Tris Gel, SYPRO Ruby and EZQ protein quantification kit are a product of Life Technologies (Molecular Probes, Invitrogen). Cell culture shake flasks were purchased from Corning. Centricon Plus-70 5000 MWCO filters were purchased from Millipore. Sequencing-grade Typsin was purchased from Promega and iTRAQ labeling reagents from AB SCIEX. CHO Cell Cultures for Secreted Protein Analysis, Standard Method

CHO DG44 cells (cGMP banked) were cultured in CD DG44 Medium supplemented with 8 mM glutamine and 0.2% pluronic (v/v). CHO-S cells (cGMP banked) were cultured in CD FortiCHO Medium supplemented with 8 mM glutamine and Anti-Clumping Agent. All cells were seeded at 3 × 105 cell/ mL in a final volume of 30 mL in a 125 mL shake flask. Cells were incubated while shaking for 3 days to a final density of (3−4) × 106 cells/mL. Cell viabilities were measured by hemocytometer to be greater than 98%. An equal number of 100 × 106 cells were removed from each culture and pelleted by centrifugation. Cell pellets were washed 3x with 5 mL of PBS. Cell pellets were brought back up in 30 mL fresh cell culture media and incubated for 8 h while shaking. Cells were again pelleted and cell culture media was filtered purified (0.2 μM). Secreted proteins in filtered supernatant were concentrated and washed (100 mM Tris pH = 8) by ultracentrifugation at 4 °C (5000 MWCO) to a final volume of 0.5−1 mL; protein concentration was measured by BCA assay (Pierce). Concentrated protein samples were digested in-solution with trypsin as follows: to 200 μg of protein, 4 μg of sequencinggrade trypsin (Promega) was added in ∼200 μL 25 mM ammonium bicarbonate and incubated for 4 h at 37 °C. Digested proteins were desalted by SPE (Strata 50um, tri-Func, C18-E), dried and redissolved in 100 μL mobile phase A (described below) for SCX separation. CHO Cell Cultures for Secreted Protein Analysis, GalNAz Click-chemistry Method

Click-iT tetra-aceylated-N-azidoacetylgalactosamine (GalNAz) was dissolved in sterile DMSO at a concentration of 100 mM. GalNAz stock solution was added to CHO DG44 and CHO-S cells to final concentration of 100 μM. Azide-acetylated sugars have been shown to have a superior cellular uptake relative to free sugars and saturate cell surface O-linked glycan modification at concentrations of 50 μM.17,22 Control DG44 and CHO-S cells were cultured with the addition of vehicle (DMSO). Cells were cultured normally as described above. After 3 days’ growth and a cell density of (3−4) × 106 cells/ mL, cell viabilities were measured to be >98% (hemocytometer); 100 × 106 cells were removed from each culture. Cells were pelleted and media supernatant filter purified (0.2 μm). Secreted proteins in filtered supernatant were concentrated and 6176



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washed (100 mM Tris pH = 8) by ultracentrifugation at 4 °C (5000 MWCO) to a final volume of 0.5−1 mL, and protein concentration was measured by BCA assay (Pierce). From this preclick-chemistry protein concentrate, 10 μL was reserved for SDS-PAGE analysis. Protein samples were stored at −80 °C for alkyne click-chemistry affinity chromatography.

iodoacetamide (30 min at room temperature). Resin was transferred to 500 μL Corning Spin-X centrifuge tube and resin was stringently washed as follows: 3× 500 μL of wash buffer; 5× 500 μL of 8 M urea; 3× 500 μL of 100 mM Tris, pH = 8; 3× 500 μL of 50% methanol/50% 100 mM Tris; 3× 500 μL of 50 mM Tris, 1 mM CaCl3, pH = 8. Bound protein was eluted by adding to the pelleted resin 200 μL containing 4 μg of sequencing-grade trypsin (Promega) dissolved in 50 mM Tris, 1 mM CaCl3. Spin columns were capped and resin was incubated at 37 °C overnight. Resin was spun and the peptide-containing flow-through reserved; hydrophobic peptides were eluted from the click-resin with 200 μL of 80% acetonitrile which was combined with the original flow-through. Eluted peptide samples were dried and purified by SPE (Strata 50um, tri-Func, C18-E) for SCX chromatography and/or iTRAQ labeling.

CHO Cell Cultures for Cytoplasmic Protein Control

CHO-S and DG44 cells were treated with GalNAz as described above. For each cell type 100 × 106 cells were pelleted and washed with PBS. Cells were resuspended in 500 μL of 100 mM Tris, pH = 8. Cells were lysed by multiple freeze thaw cycles followed by aspirating the cells through a 20 gauge needle. Cell membranes were pelleted by centrifugation and the supernatant containing the cytoplasmic proteins was filtered (0.2 μm). The final volume for each cell line was ∼0.5 mL with a protein concentration of ∼2.5 mg/mL (BCA assay). Protein samples were stored at −80 °C for alkyne click-chemistry affinity chromatography.

SDS-PAGE Verification of Click-chemistry Affinity Chromatography

Proteins were precipitated from the preclick-chemistry protein concentrate and from postclick-chemistry affinity supernatant using standard chloroform/methanol precipitation. To evaluate GalNAz incorporation and click-chemistry affinity chromatography, precipitated proteins reserved from pre- and postclickchemistry affinity were resolubilized in 50 μL 0.5% SDS and heat denatured for 10 min at 90 °C. A 2× TAMRA alkyne protein click solution was made up containing 40 μM TAMRA alkyne in 100 mM Tris (pH 8), 50% propylene glycol. 100 μL of 2× TAMRA alkyne protein click solution was added to the 50 μL protein sample along with 10 μL of 40 mM CuSO4 and 10 μL of 400 mM sodium ascorbate. The reaction was vortexed gently for 3 min before the addition of 20 μL of 100 mM Cu(I) chelating agent as described by manufacturer’s instructions. The reaction was gently vortexed for 30 min at room temperature and the TAMRA click-labeled proteins were purified by standard chloroform/methanol precipitation. To detect incorporation of azide into secreted proteins, 10 μg of TAMRA click-labeled proteins were loaded onto a NuPAGE Novex 4− 12% Bis-Tris Gel (Life Technologies). Fluorescent signals were detected using a GE Typhoon FLA 9000 fluorescent imaging laser scanner. TAMRA fluorescence (542 nm excitation and 568 nm emission) was used to monitor azide-modified proteins. To measure total protein loaded, gels were stained with SYPRO Ruby (Life Technologies) overnight and imaged following the manufacturer’s protocol (450 nm excitation and 618 nm emission) To quantify the depletion of azide-modified proteins in postclick-chemistry affinity sample, the EZQ protein quantification kit (Life Technologies) was used where the TAMRA click-chemistry signal for each protein spot was normalized by dividing the TAMRA fluorescence signal intensity by the SYPRO Ruby total protein fluorescence signal.

Alkyne Click-chemistry Affinity Chromatography for GalNAz Modified Proteins

Proteins from CHO cells treated with GalNAz were purified from either the cell culture media fraction or cytoplasmic fraction as described above. Life Technologies Click-iT protein enrichment kit containing an alkyne-modified agarose resin was used to enrich for azide-containing proteins. Click chemistry enrichment generally followed the Life Technologies protocol with a few additions. For each concentrated protein sample, 1 mL of 2× click-chemistry reaction buffer was made by combining, in the following order: 835 μL of 100 mM Tris pH 8, 20 μL of 100 mM CuSO4 (2 mM final), 125 μL of Component D (from Click-iT protein enrichment kit), and 20 μL of 1 M sodium ascorbate (20 mM final); 200 μL of 50% click-resin slurry was added to a 2 mL centrifuge tube, resin was pelleted and washed 2× with 200 μL of 100 mM Tris, pH 8. After the final wash, supernatant was removed, leaving 100 μL of alkyne click-resin. Component D is Life Technologies' propriety compound which helps to protect bio-molecules such as proteins from damage during copper catalyzed click chemistry; it can easily be found with the Click-iT reagents on the manufacturer's catalog.23 Click-chemistry affinity chromatography was done on the resin by combining 1 mL of concentrated protein sample with 1 mL of click-chemistry reaction buffer. The sample was mixed (end-overend) at room temperature for 12 h. The reaction mixture was centrifuged and the postclick-chemistry supernatant removed and reserved for SDS-PAGE analysis (described below). Resin was washed 3× with 1 mL 100 mM Tris, pH = 8. Resin was incubated with 1 mL wash buffer (100 mM Tris pH 8, 250 mM NaCl, 1% SDS, 5 mM EDTA, 10 mM of a Cu(I) chelating agent and 50 mM ascorbic acid), while shaking for 30 min; this step was repeated 2×. There is a potential correlation between copper and increased nonspecific binding to the alkyne resin. It is therefore strongly recommended to include in the wash buffer a Cu(I) chelator commonly containing pyridine or nitrile groups, even sodium cyanide can be used. Life Technologies currently does not supply a Cu(I) chelator in its Click-iT protein enrichment kit, but future (2013) versions of this kit will contain the copper chelating reagent used in this study. Bound proteins were reduced and alkylated by incubating the resin in wash buffer containing 10 mM DTT for 30 min at 60 °C followed by an additional incubation in wash buffer containing 40 mM

iTRAQ Labeling for CHO-S and DG44 Proteins from Alkyne Click-chemistry Affinity Column

Secreted protein samples from CHO-S and DG44 were processed in duplicate to demonstrate reproducibility of the quantification assay. GalNAz modified proteins were enriched by click chemistry and eluted with trypsin from the click-affinity resin as described above. Lyophilized peptides were subjected to labeling with iTRAQ 4-plex reagent (AB SCIEX). Each aliquot of peptides was dissolved in 30 μL of 0.5 M triethylammonium bicarbonate, pH 8.5 and reacted with one tube of iTRAQ reagent dissolved in 70 μL of ethanol. The reagents for each of the conditions used were: iTRAQ-114 6177



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(DG44 GalNAz secreted proteins first replicate), iTRAQ-116 (DG44 GalNAz secreted proteins second replicate), iTRAQ115 (CHO-S GalNAz secreted proteins first replicate) and iTRAQ-117 (CHO-S GalNAz secreted proteins second replicate).

calculated as 5.0% of the highest peak. Protein quantitative ratios were calculated as the median of all peptide ratios. Gene Ontology Analysis

Gene ontology (GO) terms were determined by individual analysis of identified proteins as defined by UniProt Knowledge Base (www.uniprot.org) with respect to known proteins domains, biological process and cellular component.

Strong Cation Exchange (SCX) Chromatography

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Purified peptides were separated by SCX liquid chromatography using an Agilent 1100 LC system with a Polysulfethyl A 100 × 4.6 mm, 5 μm column (Nest Group) and a flow rate of 0.25 mL/min. Mobile phase A was 10 mM sodium phosphate (Na2HPO4), 25% acetonitrile, pH = 2.8 and mobile phase B was 10 mM sodium phosphate (Na2HPO4), 25% acetonitrile, 0.4 M KCl, pH = 2.8. A linear gradient was set up as follows: 0% B from 0 to 10 min, 100% B at 70 min, 100% B at 75 min and 0% B at 80 min. Peptides were collected in 1-min fractions to give 40−60 fractions per sample. These were each desalted by ZipTips (Millipore) and dried in a SpeedVac.

RESULTS AND DISCUSSION

Enrichment of Secreted Mucin-type Glycoproteins using Click-chemistry Affinity Column

The workflow used to select and enrich for secreted mucin-type glycoproteins from cell culture media is outlined in Figure 1A.

Tandem Mass Spectrometry

The desalted and dried peptide mixtures were reconstituted in 10 μL of 0.1% Formic acid containing 5% acetonitrile. Five microliters of each enriched peptide mixture was injected onto a 100 μm x 100 mm column Atlantis dC18 employing 3 um packing (Waters Corporation) for nanoelectrospray LC−MS analysis. A flow rate of 500 nL/min was used with a gradient of 5−30% (v/v) acetonitrile in 0.1% formic acid over 60 min, 30 to 60% acetonitrile in 0.1% formic acid over additional 50 min, and then 60 to 95% acetonitrile in 0.1% formic acid over 10 min . The eluant from online nano-LC separations went directly to the Q-TOF premier instrument (Waters). The signal-to-noise intensity criterion for MS to MS/MS switch was set to 10, whereas the threshold for MS/MS to MS switch was set to above 3500 counts/s. Four component triggers were used to acquire MS/MS data with 1.4 s scan time, followed cyclically by a survey scan of 1.1 s from 400 to 1800 m/z. Ions in charge states of 2+, 3+, and 4+ detected in the survey scan were selected for MS/MS.

Figure 1. Method for the enrichment and identification of secreted mucin-type glycoproteins in CHO media. (A) Metabolic labeling of secreted proteins with GalNAz. (B) Examples of mucin-type O-linked glycans and location of GalNAz azide sugar. (C) Click-chemistry reaction with GalNAz-modified protein and alkyne affinity resin or alkyne fluorescent dye.

Database Searching and iTRAQ Protein Quantification

Raw data files from the Q-TOF instrument were processed with Mascot Distiller (Version 1.1.1.0, Matrix Science, London, U.K.) without smoothing, using charge states determined from the MS scans. All MS/MS samples were analyzed using Mascot (Matrix Science) and X! Tandem (The GPM; version 2007.01.01.1). Scaffold (Proteome Software) was used for protein identification. Scaffold performs both Mascot and X! Tandem database searching while implementing the PeptideProphet24 and ProteinProphet25 algorithms to estimate peptide and protein identification probabilities from results of databasesearch algorithms. The Swiss-Prot database for all rodents was used, assuming tryptic digestion with a fragment ion mass tolerance of 200 ppm and a parent ion tolerance of 0.5 Da. Oxidation of methionine and carbamidomethylation of cysteine were specified as variable modifications with the maximum of two missed trypsin cleavage events. MASCOT (Version 2.0) was used to quantify isobaric tag (iTRAQ) identifications. Peptides were quantified using the centroided reporter ion peak intensity. Multiple isobaric tag samples were normalized by comparing the median protein ratios for the reference channel. Protein quantitative values were derived from only uniquely assigned peptides with a minimum of two peptides per protein. The minimum quantitative value for each spectrum was

Briefly, cells were grown in the presence of GalNAz, an Nacetyl-galactosamine sugar-analogue containing an azide functional group. As demonstrated by the Bertozzi lab,17 GalNAz exploits the GalNAc salvage pathway where it is metabolized into UDP-GalNAz and transported into the Golgi to initiate mucin-type O-linked glycosylation on serine and threonine residues (Figure 1A and B). Mucin-type O-glycosylation is the most common modification of secreted proteins, therefore the metabolic incorporation of the azide group from GalNAz allowed for a reactive handle in which secreted proteins could be selectively removed from media. Glycoprotein enrichment was achieved by Cu(I) catalyzed click-chemistry by reacting the azide-containing glycoproteins in the media with our newly developed alkyne-containing agarose resin (Click-iT protein enrichment kit, Life Technologies, Figure 1C). Since azidecontaining (or GalNAz modified) proteins were attached to the agarose via a covalent triazol, the affinity resin could be stringently washed under denaturing conditions, thereby removing any contaminating or nonspecifically bound protein and allowing for a highly purified sample of secreted proteins. 6178



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Using this approach, mucin-type secreted proteins were monitored in CHO cell cultures which had been grown over a number of days; this long-term monitoring differs sharply from standard secreted protein analysis techniques, where cells are washed in PBS and incubated for only a few hours in serumfree media before analysis.16

secreted (lanes 3, 4) proteins. Both the cytoplasmic and secreted proteins from the control cells demonstrated only background TAMRA fluorescence (Figure 2B, lanes 1 and 3). Cytoplasmic proteins from GalNAz-treated cells showed a small increase in TAMRA fluorescence (Figure 2B, lane 2), while secreted proteins from GalNAz-treated cells demonstrated a strong TAMRA signal (Figure 2B, lane 4). A densitometry representation of these signal differences is shown in Figure 2C, where the total TAMRA signal of each lane was normalized to the total protein loaded by dividing by the SYPRO Ruby signal. These data demonstrate that under the conditions used in this study, only a very small number of cytoplasmic proteins were azide-modified due to GalNAz treatment. Conversely, in the cell culture supernatant a large number of proteins were seen to be modified by GalNAz, indicating preferential azide modification of secreted proteins. It was therefore assumed that epimerase conversion of GalNAz to GlcNAz was negligible and the vast majority of azide protein modification was due to GalNAz mucin-type O-linked glycosylation. This conclusion was further verified by strong cation exchange chromatography and mass spectrometry as described below.

GalNAz Modification of Proteins in Cell Culture Supernatant

The initial Bertozzi lab study on the metabolic incorporation of GalNAz demonstrated that this click-sugar was incorporated at the core position of mucin-type O-linked glycans. They also reported that any intracellular conversion of GalNAz to GlcNAz (N-azido-acetylglucosamine) by C4-epimerase was not significant relative to the direct incorporation of GlaNAz into mucin-type glycosylation.17 Zaro et al. also confirmed the intracellular metabolism of GalNAz to be preferential to mucintype glycosylation in 293T cells and did not result in other types of protein glycosylation.26 Contrary to these reports, a second study from the Bertozzi lab did demonstrate the intracellular C4-epimerase conversion of GalNAz to GlcNAz which was then incorporated into cytoplasmic O-GlcNAcylated proteins, one of the most common intracellular post-translational modifications.27 In this study, to account for this possible epimerase activity in cytoplasmic proteins, DG44 cells were grown for 3 days in media containing 100 μM GalNAz while control cells were treated with an equal volume DMSO. Cells were grown to a concentration of ∼4 × 106 cells/mL with a viability >98%. Cytoplasmic proteins were isolated as described in materials and methods. To detect azide modification in protein fractions, cytoplasmic and secreted proteins from GalNAz-treated and control cells were labeled by clickchemistry using a TAMRA-alkyne fluorescent probe and protein fractions were separated by SDS-PAGE. Azide protein modification was monitored by the TAMRA fluorescent signal, while SYPRO Ruby fluorescence was used to image total protein. As shown in Figure 2A, distinct differences in protein bands were seen between the cytoplasmic (lanes 1, 2) and the

Click-chemistry Affinity Enrichment

To demonstrate effective enrichment of metabolically labeled proteins from cell culture media, DG44 and CHO-S were cultured in media containing GalNAz and proteins from media supernatants purified as previously described. Protein fractions were subjected to click-chemistry affinity as described. To monitor the decrease or effective removal of azide-modified proteins from media supernatant samples during click-affinity chromatography, equivalent aliquots of total proteins (10 μg) were taken before and after application to the click-chemistry affinity resin and analyzed by reacting protein with a TAMRAalkyne to fluorescently label the azide-modified (GalNAz) proteins. Proteins were then separated by SDS-PAGE and changes in the TAMRA signal monitored. Figure 3 shows the TAMRA-click signal before and after enrichment, which demonstrates a significant decrease in signal after clickchemistry affinity, indicating that GalNAz modified proteins

Figure 2. SDS-PAGE of cytoplasmic and secreted protein fractions from DG44 cells treated with GalNAz. Lane 1 represents the cytoplasmic protein fraction from control (untreated) cells; lane 2 represents the cytoplasmic protein fraction from GalNAz treated cells; lane 3 represents the secreted protein fraction from control (untreated) cells and lane 4 represents the secreted protein fraction from GalNAz treated cells. Two fluorescent stains of the same gel: (A) SYPRO Ruby stain for total protein loading; (B) TAMRA-alkyne stain for the presence of GalNAz modified proteins. (C) Densitometry analysis of SDS-PAGE gel lanes, TAMRA fluorescent signal was normalized by dividing the total fluorescence by the SYPRO signal.

Figure 3. SDS-PAGE and TAMRA-alkyne click chemistry fluorescence of supernatant proteins before and after enrichment. Lanes 1−4: TAMRA-click signal in the CHO cell supernatant samples treated with GalNAz. Lanes 5 and 10: controls (non-GalNAz treated cell supernatant). Lanes 6−9: TAMRA-click signal in supernatant after GalNAz metabolically labeled proteins were selected and removed from supernatant by click-affinity chromatography. 6179



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tryptic digest. For typical MudPIT 2-D chromatography, eluted peptides were first separated by strong cation exchange chromatography (SCX) and fractions collected prior to LC− MS/MS analysis. The representative SCX chromatograms for DG44 tryptic elutions are shown in Figure 5 and demonstrate

were effectively removed or enriched from the initial media sample and remained bound to the alkyne resin. To quantify the enrichment of GalNAz-modified proteins by click-chemistry affinity, a modified version of the EZ-Q protein quantification assay (Life Technologies) was used. The protein samples taken before and after click-chemistry enrichment were reacted with TAMRA-alkyne as described above. Proteins were spotted in triplicate on the EZ-Q filter paper and the TAMRA signal measured by densitometry (Figure 4B). SYPRO Ruby

Figure 5. SCX chromatograms (215 nm) representing CHO DG44 peptides eluted form the click-chemistry affinity resin. A: media protein fraction from GalNAz-treated cells; (B) cytoplasmic protein fraction from GalNAz-treated cells; (C) media protein fraction from control (no GalNAz) cells; (D) SCX tryptic peptide standard (100 μg of BSA and β-Casein peptides).

both the ability of the click-chemistry enrichment column to select for GalNAz modified proteins, as well as the effectiveness of using trypsin to elute covalently bound proteins from the column. As expected a strong chromatographic signal was seen from the media supernatant fraction (secreted protein, Figure 5A) in which cells had been grown in the presence of GalNAz. A significantly lower signal was seen for the cytoplasmic protein fraction from the same cells (Figure 5B), indicating very small amounts of GalNAz or azide modification of intracellular proteins. This result confirms the SDS-PAGE in Figure 2 and further demonstrates that, under the conditions used in this study, azide-modified cytoplasmic proteins are not in sufficient concentration to cause the identification of false positives. The tryptic elution for the secreted protein fraction from control (not GalNAz treated) cells is show in Figure 5C and the marked difference in signal between Figure 5A and C further demonstrates the highly selective nature of this method to enrich for only GalNAz-modified or mucin-type O-glycosylated proteins with little to no background. SCX chromatography of CHO-S proteins also demonstrated identical patterns between control, secreted and cytoplasmic protein fractions (data not shown).

Figure 4. EZ-Q protein quantification of supernatant proteins before and after enrichment. Lanes 1−5: original supernatant before clickaffinity enrichment; lanes 6−10: supernatant after click-affinity enrichment; lanes 5 and 10: control supernatant (cells not treated with GalNAz). (A) SYPRO-Ruby total protein stain. (B) TAMRAalkyne click chemistry fluorescence. (C) Densitometry of TAMRA click fluorescent signal normalized to the total protein SYPRO-Ruby signal.

(Life Technologies) fluorescent stain was used to measure the total protein signal (Figure 4A). To accurately judge the decrease in GalNAz modification due to the affinity chromatography, the TAMRA fluorescence was normalized to the SYPRO Ruby total protein fluorescence (Figure 4C). The TAMRA/SYPRO ratio was seen to decrease in all samples to a level equal to the controls (not GalNAz treated), further indicating efficient removal (or enrichment) of clickable GalNAz modified proteins from both DG44 and CHO-S samples.

Mass Spectrometry Identification of Secreted and Cytoplasmic Protein Fractions in DG44 Cells

To identify DG44 secreted proteins, the collected SCX fractions (Figure 5) were analyzed by nano-LC−MS/MS. Proteins were identified by Mascot and X!-Tandem database searching using Scaffold software. Peptide identifications were accepted if they could be established at >95.0% probability as specified by the Peptide Prophet algorithm (Scaffold). Three samples were analyzed, including (1) the supernatant secreted protein fraction from cells treated with GalNAz, (2) the cytoplasmic protein fraction from cells treated with GalNAz and (3) the supernatant secreted protein fraction for control cells (not treated with GalNAz). The Venn diagrams for the number of identified proteins are shown in Figure 6. A total of

Elution of GalNAz-modified Proteins from Affinity Column

As described previously, DG44 cells were grown in media containing GalNAz, control cells were treated with an equal volume of DMSO added to the media. Secreted and cytoplasmic proteins were analyzed separately and GalNAzmodified proteins were enriched using the click-chemistry affinity resin. Since GalNAz modified-proteins were covalently bound to the affinity resin the enriched proteins were eluted by 6180



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identified with >90% confidence as predicted by Protein Prophet, which was close to 1/3 fewer proteins than identified using the GalNAz click-chemistry method (Figure 7A). A

Figure 6. Venn diagram representing total number of proteins identified from CHO DG44 cells after click affinity column enrichment. (A) Supernatant secreted protein fraction from GalNAz treated cells. (B) Supernatant secreted protein fraction from control (untreated) cells. (C) Cytoplasmic protein fraction from GalNAz treated cells.

267 proteins were identified in the secreted protein fraction; protein identifications can be seen in Supporting Information Table 1. Only a small number of proteins were identified in the cytoplasmic protein fraction and the control. These background proteins are listed in Table 1. As expected, proteins identified in the control were abundant extracellular secreted proteins, while the proteins identified in the cytoplasmic fraction were abundant intracellular proteins. These results demonstrate the ability of GalNAz click-chemistry to identify mucin-type secreted proteins, with little to no interference from nonsecreted proteins. Figure 7. Comparison of secreted proteins from CHO DG44 identified by standard method (new or fresh media incubation) and GalNAz click-chemistry method. (A) Venn diagram representing total number of proteins identified with >95% confidence. (B) Venn diagram representing the extracellular proteins (UniProtKB) identified by each method. (C) General location of proteins identified by each method (UniProtKB).

The CHO DG44 Secretome: Comparison of GalNAz Click-chemistry Method with Standard Method for Identification of Secreted Proteins

The GalNAz click-chemistry method was compared to a standard secretome proteomic method16 as described in materials and methods. The protein sample was digested with trypsin and proteins identified using MudPIT techniques as described above. Using this method, 172 proteins were

Table 1. Proteins Identified in Media Supernatant Fraction from Control (Not Treated) Cells and Cytoplasmic Fraction from GalNAz-treated Cells control cells UnitProtKB name

cellular location

Mascot score

FINC LGMN PDIA6

extracellular extracellular extracellular

137 84 77

NID1 THIO

72 50

LYAG

extracellular intra/ extracellular extracellular

PGBM

extracellular

41

41

GalNAz-treated cells name

UnitProtKB name

cellular location

Mascot score

Fibronectin Legumain Protein disulfide -isomerase A6 Nidogen-1 Thioredoxin

EF2 TPIS THIO HSP7C KPYM

intracellular intracellular intra/ extracellular intracellular intracellular

Lysosomal alpha -glucosidase HSPG

PRDX1

intracellular

60

DESM PPIA

intracellular intracellular

55 52

PRDX4

51

6PGD

intra/ extracellular intracellular

COF1 EF1G GSTM5

intracellular intracellular intracellular

43 42 42

6181

102 96 72 67 62

47

name Elongation factor 2 Triosephosphate isomerase Thioredoxin Heat shock cognate 71 Pyruvate kinase isozymes M1/ M2 Peroxiredoxin-1 Desmin Peptidyl-prolyl cis−trans isomerase A Peroxiredoxin-4 6-phosphogluconate dehydrogenase Cofilin-1 Elongation factor 1-gamma Glutathione S- transferase Mu 5



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Table 2. Top 20 Mascot Scoring Proteins Identified by Standard Method (New or Fresh Media Incubation) and GalNAz Clickchemistry Method standard UnitProtKB name

cellular location

Mascot score

ACTG HS90A

intracellular intracellular

331 295

HSP7C

intracellular

287

HS90B TSP1 GRP78

intracellular extracellular intracellular

284 282 226

ACTA A4 CSPG4

intracellular extracellular extracellular

216 210 193

KPYM

intracellular

180

G3P

intracellular

157

CLUS

extracellular

156

ALDOA

intracellular

153

ANXA2 CSPG4

extracellular extracellular

144 139

VIME

intracellular

LEG1 NID1 ENOA UBIQ

extracellular extracellular intracellular intracellular

GalNAz click-chemistry name Actin, cytoplasmic Heat shock protein HSP 90alpha Heat shock cognate 71 kDa protein Heat shock protein HSP 90-beta Thrombospondin-1 78 kDa glucose-regulated protein Actin, aortic smooth muscle Amyloid beta A4 protein Chondroitin sulfate proteoglycan 4 Pyruvate kinase isozymes M1/ M2 Glyceraldehyde-3-phosphate dehyd Clusterin

UnitProtKB name

cellular location

Mascot score

GPC1 CSPG4


3017 2129

NID1

extracellular

1905

Glypican-1 Chondroitin sulfate proteoglycan 4 Nidogen-1

PGBM CATB LAMB1


1686 1004 1004

Basement membrane-specific Cathepsin B Laminin subunit beta-1

LGMN A4 FINC


1000 969 866

Legumain Amyloid beta A4 protein Fibronectin

PXDN

extracellular

764

Peroxidasin homologue

CLUS

extracellular

676

Clusterin

LDLR

extracellular

514

LGMN

extracellular

483

Low-density lipoprotein receptor Legumain

name

PDIA6 TSP1


463 434

Protein disulfide-isomerase A6 Thrombospondin-1

137

Fructose-bisphosphate aldolase A Annexin A2 Chondroitin sulfate proteoglycan 4 Vimentin

LTBP1

extracellular

410

135 129 128 118

Galectin-1 Nidogen-1 Alpha-enolase Ubiquitin

SPRC DAG1 APLP2 MAMC2

extracellular extracellular extracellular extracellular

404 379 375 370

Latent-transforming growth factor SPARC Dystroglycan Amyloid-like protein 2 MAM domain-containing protein 2

significant overlap of extracellular proteins were identified using the two methods (Figure 7B), which demonstrated the GalNAz method was not only inclusive of extracellular proteins from the standard method but also increased that number by greater than 70%. The modest 22% overlap of total proteins identified using the two methods (Figure 7A), can be explained by looking at the cellular location of identified proteins. Figure 7C shows that the majority of proteins identified by the GalNAz click-chemistry method were extracellular in location. Conversely, the majority of proteins identified by the standard method were intracellular. This large number of nonsecreted proteins demonstrated that the PBS washes and new or fresh media incubation still allowed for significant contamination from abundant intracellular proteins that tend to accumulate during sample processing and most likely lead to the identification of false positives. The difference between the quality of the protein data identified by the two methods was further highlighted by comparing the top 20 proteins (as dictated by the top 20 Mascot M-scores) from each method (Table 2). For the standard method, 12 of the top 20 proteins were intracellular, which included highly abundant actin and heat shock proteins; only 8 of these proteins were extracellular. In contrast, all of the top 20 proteins identified by the GalNAz method were extracellular. The first intracellular proteins identified using the GalNAz method were seen beyond the top 40 highest scoring proteins, which were heat shock protein 71 at number 46 and pyruvate kinase isozymes at number 47. Furthermore, the

Mascot scores for the GalNAz method shown in Table 2B are significantly higher, likely a direct result of the accumulation of secreted proteins over 3 days and explains the greater number of overall proteins identified by this method. To further emphasize the quality of the secretome proteomics data achieved from the GalNAz click-chemistry method, Figure 8 gives a summarized Gene Ontology comparison (as defined by the UniProt knowledge base). Protein domains are shown in Figure 8A. The signal domain was the most common identified by the GalNAz method (>40% of all proteins). This domain is indicative of the secretory pathway since it codes for the hydrophobic signal peptide that initiates secretion by inserting into the ER during protein synthesis. In contrast, no domain was most common for the standard method (>30%), indicating intracellular or cytoplasmic proteins. Cellular components or protein locations are shown in Figure 8B. Secreted was the most common location identified by the GalNAz method (∼40%); membrane or membrane-associated proteins were also common (∼25%). Again, in contrast, the standard method had a tendency to identify proteins that were either cytoplasmic or nuclear. Figure 8C shows the biological process or function for identified proteins. Here the standard method identified more proteins that had functions commonly found intracellularly, such as chaperones, nucleic acid binding, structural or biosynthesis. The GalNAz method tended to identify proteins with functions that are typical of secreted proteins, such as cell adhesion, 6182



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Figure 9. Venn diagram of proteins secreted proteins identified from CHO-S and DG44 cells.

secretomes is given in Supporting Information Tables 1 and 2, respectively. This is the most comprehensive list of secreted proteins currently identified in CHO cell culture. Quantification of DG44 and CHO-S Secretome

To quantify changes between the secretome of DG44 and CHO-S cells, peptides from tryptic digests were labeled with iTRAQ 4-plex isobaric tags. To validate and demonstrate reproducibility of the assay, DG44 and CHO-S cell types were processed in duplicate. Two cultures each of DG44 and CHO-S were processed using the GalNAz click-chemistry method as described above. The two DG44 samples were labeled with iTRAQ isotopic labels m/z 114 and 116. The two CHO-S samples were labeled with iTRAQ isotopic labels m/z 115 and 117. Labeled peptides were combined and analyzed by MudPIT 2D-LC−MSMS. A total of 325 proteins were identified with greater than 95% confidence and relative protein quantification was calculated by Mascot. The duplicate DG44 and CHO-S samples demonstrated good correlation in concentrations changes. Table 3 lists a total of 35 secreted proteins whose concentration changes were 2-fold or greater between DG44 and CHO-S. Relative concentration changes are represented by the heat map in Table 3, where green corresponds to low abundance and red to high abundance. Of these proteins, 28 were extracellular, with the majority being either secreted or membrane associated. The most dramatic relative concentration changes were seen in the proteins: biglycan, collagen alpha-1(VI) chain, matrix metalloproteinase-19, procollagenlysine 2-oxoglutarate 5-dioxygenase 1 and SWI/SNF complex subunit SMARCC2. It has been generally assumed that the secreted proteins or HCP from CHO cells are essentially the same;15 the results shown in Table 3 contradict this assumption. Understanding the relevant HCP is important for the design of therapeutic protein purification processes, where differences in HCP between cell lines may dictate changes in the purification conditions used. This knowledge is also important for the optimization of the cell culture environment. It is unknown which, if any, secreted proteins play a positive or detrimental role toward cell growth, viability or recombinant protein titer, but this study takes the first step to identifying these key components.

Figure 8. Gene Ontology (UnitProtKB) comparison of secreted proteins identified by GalNAz click-chemistry method and standard method (new or fresh media incubation). (A) Protein domains; (B) cellular component/protein location; (C) protein biological process.

extracellular matrix, cell growth, proteases and protease inhibitors. The CHO-S Secretome

To identify CHO-S secreted proteins using the GalNAz clickchemistry method, cells were cultured in the presence of GalNAz and secreted proteins enriched using the clickchemistry affinity chromatography as described above. Tryptic peptides were fractionated by SCX and analyzed by nano-LC− MS/MS, as described above. Results were compared with the previously identified DG44 secretome. For CHO-S cells, 256 proteins were identified with 95% confidence (Figure 9). Similar to the DG44 cells, the majority of proteins identified were extracellular, either being secreted or membrane associated. A total of 171 proteins (∼ 70%) were identified in both DG44 and CHO-S cell types, demonstrating a significant overlap in the secretome of these cell lines. A complete list of the identified CHO DG44 and CHO-S

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CONCLUSIONS The analysis of secreted proteins is challenging. To avoid contamination from nonsecreted proteins, cells are typically incubated in new or fresh media for a few hours before analysis. This short incubation time significantly dilutes the sample, decreasing the identification of low abundance secreted proteins. It also limits proteomic monitoring to one time point during the culture’s life, generally at a maximum cell density. To improve the analysis of secreted proteins, we cultured CHO cells in media containing N-azido-galactosamine 6183



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Table 3. iTRAQ Quantification of DG44 and CHO-S Secretomea

a

Heat map represents secreted protein concentrations with fold changes greater than 2, where red designates a high relative concentration and green a low relative concentration.

enzymes in the trans Golgi network, these proteins are tagged for delivery only to the lysosome. These enzymes can escape this normal packaging process and be transported by the default secretory pathway to the cell surface, where they are secreted.29 Significant evidence also suggests that lysosomes are not just an end point for endocytosis but can also function as secretory organelles, where they have the ability to fuse with the plasma membrane allowing cells to eliminate indigestible debris or to secrete proteins.29−31 The majority of cells studied with known secretory lysosomes are immune cells, but the use of lysosomes as secretory organelles is likely more widespread and has been documented in fibroblasts and epithelial cells,31−33 implying

(GalNAz), which was metabolically incorporated into mucintype O-linked glycans of secreted proteins. These proteins were effectively enriched using click-chemistry from the cell culture media, allowing for the analysis of secreted proteins after days of cell growth. Interestingly the CHO-S and DG44 secretomes identified in this study contained a significant number of proteins associated with lysosomes and melanosomes. The interaction or relationship between lysosomes, melanosomes, endosomes and the secretory pathway is highly dynamic, involving the location of secretory cargos that is not necessarily clearly defined28,29 For example, during the production of lysosomal hydrolase 6184



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dimensional liquid chromatography−tandem mass spectrometry

that lysosomal fusion with the plasma membrane may be an ubiquitous form of exocytosis. The presence of lysosomal proteins found as part of the CHO secretome in this study tends to indicate that CHO cells, which are a type of epithelial cell, also contain secretory lysosomes. Although it may be argued that the presence of lysosomes in cell culture media results from cell death, the measured viability of the CHO cells in this study was extremely high (98−100%) and the abundance of lysosomal proteins identified was far greater than what would be expected from such high viabilities. The function of secretory lysosomes in CHO cells is unknown and may be indicative of cell stress, but could also be a sign of rapid growth and proliferation. The DG44 and CHO-S proteins identified in this study along with this click-chemistry methodology can be used to better understand the dynamic extracellular environment of these important production cell lines. It is our hope that this and future studies will help us improve essential cell culture conditions and develop new chemically defined media formulations. It should be noted that this study was focused on establishing a baseline of secreted proteins specific to CHO cell culture; our future research which will focus on changing the culture conditions to bioreactor or fed batch cell culture typically used during therapeutic drug production. This GalNAz click-chemistry technology is ideal for these types of studies since secreted proteins can be monitored over extended time periods. Finally, these studies used chemically defined media (CD FortiCHO Medium and CD DG44 Medium) and it was beyond the scope of this project to examine other cell culture media conditions. Our future research should therefore extend this technology to cells grown with serum supplementation and demonstrate the usefulness of this secretome monitoring technique to be used on cell types that require serum for growth.

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(1) Kim, J. Y.; Kim, Y. G.; Lee, G. M. CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl. Microbiol. Biotechnol. 2012, 93 (3), 917−30. (2) Jayapal, K. P.; Wlaschin, K. F.; Hu, W. S.; Yap, M. G. Recombinant protein therapeutics from CHO cells20 years and counting. Chem. Eng. Prog. 2007, 103, 40−47. (3) Gupta, P.; Lee, K. H. Genomics and proteomics in process development: opportunities and challenges. Trends Biotechnol. 2007, 25 (7), 324−30. (4) Urlaub, G.; Chasin, L. A. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. U.S.A. 1980, 77 (7), 4216−20. (5) Urlaub, G.; Kas, E.; Carothers, A. M.; Chasin, L. A. Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell 1983, 33 (2), 405−12. (6) Wurm, F. M. et al. Methotrexate and CHO cells: productivity and genetics of amplified expression vector sequences. In Production of Biologicals from Animal Cells in Culture; Butterworths: Oxford, 1990. (7) Wurm, F. M.; Hacker, D. First CHO Genome. Nat. Biotechnol. 2011, 29 (8), 718−20. (8) Puck, T. T.; Ciecirua, S. J.; Robinson, A. Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects. J. Exp. Med. 1958, 108 (6), 945−956. (9) Deaven, L. L.; Petersen, D. F. The chromosomes of CHO, an aneuploid Chinese hamster cell line: G-band, C-band, and autoradiographic analyses. Chromosoma 1973, 41, 129−44. (10) Xu, X.; Nagarajan, H.; Lewis, N. E.; Pan, S.; Cai, Z.; Liu, X.; Chen, W.; Xie, M.; Wang, W.; Hammond, S.; Andersen, M. R.; Neff, N.; Passarelli, B.; Koh, W.; Fan, H. C.; Wang, J.; Gui, Y.; Lee, K. H.; Betenbaugh, M. J.; Quake, S. R.; Famili, I.; Palsson, B. O. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat. Biotechnol. 2011, 29 (8), 735−41. (11) Wlaschin, K. F.; Seth, G.; Hu, W. S. Toward genomic cell culture engineering. Cytotechnology 2006, 50 (1−3), 121−40. (12) Wang, X.; Hunter, A. K.; Mozier, N. M. Host cell proteins in biologics development: Identification, quantitation and risk assessment. Biotechnol. Bioeng. 2009, 103 (3), 446−58. (13) Shukla, A. A.; Jiang, C.; Ma, J.; Rubacha, M.; Flansburg, L.; Lee, S. S. Demonstration of robust host cell protein clearance in biopharmaceutical downstream processes. Biotechnol. Prog. 2008, 24 (3), 615−22. (14) Liu, N.; Brevnov, M.; Furtado, M.; Liu, J. Host Cellular Protein Quantification. Bioprocess Int. 2012, 10 (2), 44−50. (15) Krawitz, D. C.; Forrest, W.; Moreno, G. T.; Kittleson, J.; Champion, K. M. Proteomic studies support the use of multi-product immunoassays to monitor host cell protein impurities. Proteomics 2006, 6 (1), 94−110. (16) Dowling, P.; Clynes, M. Conditioned media from cell lines: a complementary model to clinical specimens for the discovery of disease-specific biomarkers. Proteomics 2011, 11 (4), 794−804. (17) Hang, H. C.; Yu, C.; Kato, D. L.; Bertozzi, C. R. A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (25), 14846−51. (18) Dube, D. H.; Prescher, J. A.; Quang, C. N.; Bertozzi, C. R. Probing mucin-type O-linked glycosylation in living animals. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (13), 4819−24. (19) Hart, G. W.; Haltiwanger, R. S.; Holt, G. D.; Kelly, W. G. Glycosylation in the nucleus and cytoplasm. Annu. Rev. Biochem. 1989, 58, 841−74. (20) Varki, A. C., R.D.; Esko, J. D.; Freeze, H. H.; Stanley, P.; Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Essentials of Glycobiology, 2nd ed.; Cold Spring Harbor Laboratory Press: La Jolla, CA, 2009. (21) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.;

ASSOCIATED CONTENT

S Supporting Information *

Two supplementary tables are provided that include all secreted proteins identified from DG44 and CHO-S cell cultures using the GalNAz click-chemistry method. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Life Technologies, 29851 Willow Creek Rd, Eugene, OR 97401. E-mail: [email protected]. Phone: 406-546-3062. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge Graziella Piras and Michelle Sabourin for their help with CHO cell culture conditions and Brian Agnew, Tamara Nyberg and Robert Aggeler for their help and advice on click-chemistry and metabolic labeling.

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ABBREVIATIONS CHO cells, Chinese hamster ovary cells; GalNAz, N-azidogalactosamine; GlcNAz, N-azido-glucosamine; MudPIT, multidimensional protein identification; 2D-LC−MS/MS, two6185



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Jacobson, A.; Pappin, D. J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 2004, 3 (12), 1154−69. (22) Jacobs, C. L.; Yarema, K. J.; Mahal, L. K.; Nauman, D. A.; Charters, N. W.; Bertozzi, C. R. Metabolic labeling of glycoproteins with chemical tags through unnatural sialic acid biosynthesis. Methods Enzymol. 2000, 327, 260−75. (23) Uttamapinant, C.; Tangpeerachaikul, A.; Grecian, S.; Clarke, S.; Singh, U.; Slade, P.; Gee, K. R.; Ting, A. Y. Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling. Angew Chem Int Ed Engl 2012, 51 (24), 5852−5856. (24) Keller, A.; Nesvizhskii, A. I.; Kolker, E.; Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 2002, 74 (20), 5383−92. (25) Nesvizhskii, A. I.; Keller, A.; Kolker, E.; Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 2003, 75 (17), 4646−58. (26) Zaro, B. W.; Bateman, L. A.; Pratt, M. R. Robust in-gel fluorescence detection of mucin-type O-linked glycosylation. Bioorg. Med. Chem. Lett. 2011, 21 (17), 5062−6. (27) Boyce, M.; Carrico, I. S.; Ganguli, A. S.; Yu, S. H.; Hangauer, M. J.; Hubbard, S. C.; Kohler, J. J.; Bertozzi, C. R. Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (8), 3141−6. (28) Raposo, G.; Marks, M. S. Melanosomes–dark organelles enlighten endosomal membrane transport. Nat. Rev. Mol. Cell Biol. 2007, 8 (10), 786−97. (29) Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. Transport from the Trans Golgi Network to Lysosomes. In Molecular Biology of the Cell, 4th ed.; Garland Science: New York, 2002. (30) Holt, O. J.; Gallo, F.; Griffiths, G. M. Regulating secretory lysosomes. J. Biochem. 2006, 140 (1), 7−12. (31) Stinchcombe, J.; Bossi, G.; Griffiths, G. M. Linking albinism and immunity: the secrets of secretory lysosomes. Science 2004, 305 (5680), 55−9. (32) Rodriguez, A.; Webster, P.; Ortego, J.; Andrews, N. W. Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells. J. Cell Biol. 1997, 137, 93−104. (33) Jaiswal, J. K.; Andrews, N. W.; Simon, S. M. Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells. J. Cell Biol. 2002, 159, 625.

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Identifying the CHO Secretome using Mucin-type O-Linked

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