Studying O-Linked Protein Glycosylations in Human Plasma Taufika Islam Williams, Diana A. Saggese, and David C. Muddiman* W. M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695 Received January 25, 2008
Abstract: Recent investigations have implicated aberrant glycosylations in various malignancies, including epithelial ovarian cancer (EOC). The protocol here identifies O-linked carbohydrate patterns in EOC plasma glycoproteins through chemical cleavage and purification of these glycans. Dialyzed plasma is subjected to reductive βelimination with alkaline borohydride to release O-linked oligosaccharides from glycoproteins. Enrichment of released glycans, as well as removal of peptide and other contaminants, is followed by carbohydrate pattern analysis with MALDI-FT-ICR-MS. Keywords: protein O-glycosylations • plasma • epithelial ovarian cancer (EOC) • matrix-assisted laser desorption/ iozation (MALDI) • Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR-MS)
Introduction The field of proteomics in general and glycomics in particular is increasingly being driven by the development of specialized, robust and reproducible methodologies for the interrogation of complex matrices like blood and cerebrospinal fluid. The benefits of biomarker discovery in the glycomics arena are several-fold, due in no small part to the structural complexity and diversity of carbohydrates, their important roles in cellular processes and because glycoproteins describe a more specific target analyte as a subset of the entire proteome. Over half of all translation products are glycosylated, with carbohydrate volume often being significantly greater than that of the glycoprotein anchor. Protein glycosylations influence an array of cellular processes, ranging from protein folding, stability, cell-cell adhesion, cellular signaling pathways and overall maintenance of homeostasis. Aberrant carbohydrate profiles in glycoproteins were first linked to cancer in the late 1970s.1 Since then, mounting evidence has demonstrated that differences in N- and O-linked glycosylations do correspond with increasing tumor burden and poor prognosis in a variety of malignancies.2,3 Specifically, recent studies with blood products, cellular secretions, and cancer cell surfaces have established a clear link between abnormal glycosylation profiles and disease progression in epithelial ovarian cancer (EOC).3–6 Glycans were implicated in EOC in the early days of biomarker discovery for this disease, when Gehrke and co-workers7 * Corresponding author. David C. Muddiman, Professor of Chemistry, W. M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204. Tel, (919) 513-0084; fax, (919) 513-7993; e-mail,
[email protected].
2562 Journal of Proteome Research 2008, 7, 2562–2568 Published on Web 04/19/2008
quantified carbohydrates in serum proteins from cancer patients by gas-liquid chromatography (GLC). Since then, investigations have documented the influence of aberrant glycan patterns in the development2,8 and metastasis4,9 of this gynecologic malignancy, thereby highlighting their potential value in diagnosis,3 prognosis,3,10 and treatment.5 The highly Oglycosylated mucin CA125,11,12 the standard diagnostic marker for EOC, lacks the necessary sensitivity and specificity for use as a population screening biomarker. O-Glycosylations have recently been studied by Lebrilla and colleagues in EOC13,14 and breast cancer15 sera and cell lines. In this approach, glycans were selectively cleaved from glycoproteins by reductive βelimination, a strategy exploited in a number of protocols for the O-glycan analysis of other systems.16–20 Laboratory-to-laboratory reproducibility issues, especially in the analysis of a complex matrix like plasma, call for wellestablished and robust protocols. While O-glycan analysis of glycoproteins through reductive β-elimination has been reported several times in the literature, a detailed and reproducible methodology with which to interrogate highly complex and precious biological samples is not available. Blood plasma, for instance, consists of a large number of proteins, electrolytes, nutrients, hormones, clotting factors, vitamins and waste products, spanning a wide dynamic range. To analyze any component of this biofluid calls for the careful consideration of all components, their interactions with one another and with the tools of the analytical method to be employed. Specifically, in a comprehensive approach to O-glycan studies of plasma glycoproteins, there are numerous, time-consuming steps involved and each step features an array of variables that require individual optimization. We have performed an exhaustive study of these variables and, in conjunction with material already available in the literature, arrived at a protocol that is effective and reproducible. The work presented here details a protocol for O-glycan investigations in human plasma. We would like to point out that this communication does not describe a novel approach to carbohydrate profiling but a refinement of existing protocols for the analysis of biofluids, with a stepwise examination of different experimental stages. The effectiveness and major points in the optimization of the methodology are detailed, with close attention given to purification of released glycans through solid-phase extraction (SPE),21 exhaustive pronase digestion of peptide contaminants22–25 and drop dialysis.26 Plasma glycoprotein enrichment by lectin chromatography27–30 prior to chemical release of O-glycans was also explored. An evaluation of O-glycan distribution in a sample of late-stage EOC plasma with an appropriate age- and menopause-matched control is 10.1021/pr800066e CCC: $40.75
2008 American Chemical Society
technical notes
O-Linked Protein Glycosylations in Plasma
Table 1. Experimental Conditions Explored for the Purification and Enrichment of Carbohydrates Released from Plasma O-Glycoproteins by Reductive β-Elimination experimenta
parameter spaceb
Lectin chromatography for O-linked glycoprotein enrichment prior to β-elimination29
agarose-bound jacalin column
Exhaustive pronase digestion of released glycans to remove protein contaminants
enzyme buffers
enzyme concentration
enzyme digestion time
enzyme quenching Drop dialysis for additional glycan purification to remove amino acids, peptides, salts, etc
dialysis time
number of dialysis steps sample volume
Gains in sample cleanup did not offset sample loss as a result of additional chromatography steps
• 20 µL • 10 µL • 5 µL
e10 µL was appropriate for glycan purification. Higher volumes were not dialyzed adequately
a Optimization of MALDI-FT-ICR sample preparation and analysis31 described elsewhere. stated: • plasma dialysis with Slide-A-Lyzer cassettes with no lectin chromatography step • 50 mM NH4HCO3 pronase buffer • 5 mg/mL pronase • 4 h digestion time • pronase quenched by boiling for 2 min post-digestion • 2 h drop dialysis in a single step • 10 µL drop dialysis volume.
provided. Differential glycan distribution between control and disease states was observed upon analysis by MALDI-FT-ICR mass spectrometry.
Experimental Section Please refer to Supporting Information for a detailed experimental procedure for O-glycan analysis of plasma glycoproteins. While the optimization of this protocol involved the exploration of a large parameter space (Table 1), the final, optimized procedure is given here. Materials. Mucin type III from porcine stomach, trifluoroacetic acid (TFA), sodium borohydride (NaBH4), sodium hydroxide (NaOH), sodium chloride (NaCl), hydrochloric acid (HCl), 2,5-dihydroxybenzoic acid (2,5-DHB), and pronase were purchased from Sigma (St. Louis, MO). Ammonium bicarbonate (NH4HCO3) came from Fisher (Hampton, NH). HPLC-grade water and acetonitrile (ACN) were obtained from VWR International (Suwanee, GA). Graphitized carbon solid-phase extraction (SPE) cartridges came from Alltech (Deerfield, IL) and drop dialysis membranes from Millipore (Burlington, MA). Slide-ALyzer dialysis cassettes, MWCO 7000, were purchased from Pierce (Rockford, IL). Plasma Dialysis for Low MW Contaminant Removal and Desalting. Twelve 100 µL aliquots of human plasma were obtained from the same source (Red Cross). A 1 mg/mL aq
outcome
• lectin chromatography of plasma followed by dialysis • plasma dialysis with no chromatographic enrichment • 50 mM NH4HCO3 • 5 mM CaCl2 with 50 mM NH4HCO3 • no digestion • 1 mg/mL • 5 mg/mL • 10 mg/mL • 15 mg/mL • no digestion •1h •4h •6h • 10 h • no quenching • boiling for 2 min • 0 min • 30 min • 1.0 h •2h • 2.5 h •3h • one 2 h step • two 1 h steps
b
Presence of calcium ions to protect from autolysis did not improve data 5 mg/mL of pronase was appropriate for exhaustive digestion Exhaustive digestion with pronase for 4 h was sufficient
Deactivating the pronase improved the data Dialyzing glycan elutions for 2 h was sufficient. Longer dialysis times resulted in glycan loss
One dialysis step was appropriate for glycan purification
Key experimental parameters held constant unless otherwise
mucin internal standard solution was prepared and 100 µL of this solution was added to each of six of the plasma samples. To the other six samples, 100 µL of nanopure water was added. A blank (200 µL nanopure water) and a positive control (100 µL mucin solution + 100 µL nanopure water) were also prepared. With Pierce Slide-A-Lyzer dialysis cassettes, all 14 samples were dialyzed against 300 mL of nanopure water for 24 h. The samples were then recovered and lyophilized to dryness. Reductive β-Elimination of O-Glycans in Glycoproteins. Approximately 4 mg of lyophilized plasma (with or without mucin) was weighed and transferred into separate 15 mL polypropylene tubes for reductive β-elimination. An alkaline borohydride solution (1 M NaBH4 and 0.1 M NaOH) was then prepared and 400 µL was added to each sample. The same volume of basic reagent was added directly to the lyophilized control samples, vortexed, and transferred to separate 15 mL polypropylene tubes. The 14 samples were then incubated in a heating block for 16 h at 42 °C. Following the reaction, the sample tubes were placed on ice and 1 M HCl was added dropwise to each to quench the reaction. Addition of acid was continued until the pH was between 3 and 5. The solutions were subsequently vortexed and subjected to solid-phase extraction for oligosaccharide purification and enrichment. Journal of Proteome Research • Vol. 7, No. 6, 2008 2563
technical notes Solid-Phase Extraction (SPE) of O-Glycans. Solid-phase extraction was performed using graphitized carbon cartridges. Prior to SPE, the cartridges were preconditioned with 2 column vol of nanopure water, followed by 1 column vol of 80% ACN in 0.05% aq TFA (v/v) and finally 2 more column vol of nanopure water. In the loading phase of SPE, sample solution from the β-elimination process was added to the SPE cartridge and allowed to pass through without external pressure. In the desalting phase, the cartridge was washed with approximately 50 mL of nanopure water. Elution (∼2 mL/min) from the cartridge was carried out in a stepwise manner using 10% ACN (v/v; elution of small oligosaccharides), 20% ACN (v/v; elution of large oligosaccharides) and 40% ACN in 0.05% aq TFA (v/v; elution of acidic oligosaccharides). The fractions were evaporated to dryness and each was reconstituted in 40 µL of nanopure water. Protein/peptide contaminants in the samples were then removed by protease digestion. Exhaustive Pronase Digestion of Peptide Contaminants. A 5 mg/mL pronase solution in 50 mM aq NH4HCO3 was prepared and combined in a 1:1 ratio with sample elutions from the SPE step. Sample/enzyme solutions were then placed on a heating block and digested at 37 °C for 4 h to digest protein/ peptide contaminants. Following digestion, the solutions were boiled in a water bath at 100 °C for 2 min to quench the enzyme. Additional glycan purification was then undertaken by drop dialysis. Drop Dialysis for Glycan Purification. Separate Millipore V-series membranes floating in 100 mL of nanopure water were used to dialyze the digested samples. A small volume (∼10 µL) of each enzyme/sample mixture was pipetted onto separate membranes, placing the drop at the center. Dialysis was performed for 2 h. The sample drops were then removed and processed for MALDI-FT-ICR-MS analysis. MALDI-FT-ICR-MS Sample Preparation and Analysis. Purified glycan samples were combined in a 1:1 ratio with 2,5-DHB matrix solution. For positive ion MALDI, the matrix solution was prepared by dissolving 100 mg of 2,5-DHB in 1 mL of 1:1 ACN/50 mM NaCl. An equivalent mass of matrix was dissolved in 1 mL of 1:1 ACN/H2O for experiments in the negative ion mode. Analyte/matrix solutions were then vortexed and spinned down. A 0.8 µL aliquot of the sample/matrix mixture was spotted on an Applied Biosystems (Foster City, CA) MALDI target (Part no. 433375). It is preferable to spot in triplicate. Samples were dried on-target with a cold stream of air and subjected to MALDI-FT-ICR-MS analyses.31 MALDI-FT-ICR-MS. Mass spectrometric data were obtained with a FT-ICR mass spectrometer interfaced to a ProMALDI ion source (Varian, Lake Forest, CA). A Nd:YAG frequency tripled-laser (355 nm) promoted desorption/ionization of analyte. The Varian Omega 2XP data station was used for all processing and signal generation and the standard broadband pulse sequence was employed. The instrument features a 9.4 T horizontal bore superconducting magnet with a 128 mm bore (Cryomagnetics, Inc. Oakridge, TN). For the study of the entire mass range of released O-glycans, mass spectrometric data was acquired with the MALDI-FT-ICR quadrupole ion guide optimized at m/z 400 and 900. O-Glycan Analyses of EOC Plasma Glycoproteins. An EOC plasma sample from a subject with advanced disease (Stage IV) and an appropriate age- and menopause-matched control (cystadenoma) was subjected to the procedure outlined here to observe any differences in glycan patterns between cancerous and normal states. 2564
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Williams et al.
Figure 1. MALDI-FT-ICR mass spectra of O-glycans obtained through (a) a plasma processing method involving no lectin chromatography step and (b) a procedure involving lectin chromatography of plasma with agarose-bound jacalin. Peaks identified belong to HexA series 1 (green asterisks) and 3 (red asterisks).
Results and Discussion All MS data reported here are from glycan elution with 10% ACN and the FT-ICR ion guide optimized at m/z 400 (positive ion mode). Important points in the optimization of the procedure for the analysis of O-glycans in plasma glycoproteins are provided in Table 1. These experiments identified key parameters that influence glycan analysis. A majority of the problems that may arise in the procedure can be traced to one of the sample cleanup steps. In general, adequate desalting (∼50 mL nanopure water in SPE) and elimination of contaminating peptides can yield the desired plasma glycan MS data. Enrichment techniques were investigated for incorporation both prior to and following reductive β-elimination. While lectin chromatography has been previously applied in the enrichment of plasma glycoproteins,27–30 we did not observe any significant benefit to incorporating it into the strategy described here (prior to chemical cleavage of glycans; see Supporting Information). Agarose-bound jacalin was selected as the lectin of choice due to known affinity to glycoproteins in general, with some preference for O-linked glycoproteins (personal communication with Dr. William S. Hancock, Northeastern University, Boston, MA, and Vector Laboratories, Burlingame, CA).29 Indeed, gains in sample enrichment did not offset sample loss upon incorporation of these additional (chromatography) steps in the procedure, as shown in Figure 1. Plasma glycan peaks identified in the mass spectrasprimarily series of hexuronic acid (HexA) separated by 198 Da (HexA + Na - H)sare given in Table 2. O-Glycans were identified by accurate mass, comparison with previously published results and the OsCal program from the Lebrilla laboratory (UC Davis, Davis, CA).13 Exchange of -COOH protons with Na+ from the NaCl dopant produced satellite peaks 22 mass units apart. HexA monomers (mainly of glucuronic acid) and oligomers have been reported in human sera.13,15,32,33 Solid-phase extraction of released glycans with graphitized carbon columns was well-suited for the enrichment of carbohydrates released from glycoproteins by reductive β-elimination. The stationary phase allowed free passage of salts while binding strongly with peptides and proteins, as well as to a
O-Linked Protein Glycosylations in Plasma Table 2. Series of Sodium-Adducted Hexuronic Acid (HexA) Oligosaccharides Found in Human Plasma series no
observed m/z
oligosaccharide compositiond
1a
616.968 814.965 1012.978 1211.008 1409.011 559.015 757.021 551.015 749.005 946.998 1145.051 1343.033 1541.076 554.990 752.994 950.996 1149.003
[HexA]3 [HexA]4 [HexA]5 [HexA]6 [HexA]7 361 + [HexA]1 361 + [HexA]2 unknown 551 + [HexA]1 551 + [HexA]2 551 + [HexA]3 551 + [HexA]4 551 + [HexA]5 unknown 555 + [HexA]1 551 + [HexA]2 551 + [HexA]3
2b 3c
4b
a Series 1 peaks in preceding mass spectra (green asterisks). Observed in very low ion abundances by MALDI-FT-ICR-MS. c Series 3 peaks in preceding mass spectra (red asteriks). d Identification through accurate mass, previously published results and the OsCal program from the Lebrilla laboratory.13 b
lesser extent with glycans. Solvents with increasing organic content eluted O-glycans, with the proteins remaining captured. MS analysis of the SPE elutions without further purification showed a host of nonglycan peaks, many of which were protein contaminants from plasma that were not successfully retained by the SPE cartridge. Exhaustive digestion of glycan samples with an indiscriminate protease such as pronase, followed by dialysis to remove amino acids, low molecular weight peptides and other contaminants, significantly improved oligosaccharide ion abundance in the mass spectra. Several pronase concentrations were examined, along with different buffers, digestion times, as well as enzyme quenching (Table 1). Our results indicated that a 4 h digestion with a 5 mg/mL pronase solution in 50 mM NH4HCO3, followed by enzyme quenching by boiling for 2 min, was appropriate for glycan investigations. Several parameters were also explored for drop dialysis to remove low MW contaminants. These included dialysis time, number of dialysis steps (each with a fresh membrane), as well as sample volume. The quality of oligosaccharide data was closely associated with dialysis time; glycan ion abundance increased with dialysis time up to a certain point, beyond which longer times resulted in the loss of glycan abundance (Figure 2). As shown in Figure 2, a dialysis time of 2 h produced maximum ion abundance from O-glycans while minimizing ion abundance from peptides and other contaminants. In addition, the appearance and crystal definition of MALDI sample spots improved dramatically with longer dialysis times. One 2 h drop dialysis step with sample volume not exceeding 10 µL was adequate for oligosaccharide profiling. Inorganic salts are readily removed from the sample droplet in drop dialysis. Allmaier and colleages26 have evaluated the dialysis membrane used here for glycoprotein purification. While the manufacturer correlates pore size (25 nm) to a molecular weight cutoff of 20 kDa, the authors reported the successful dialysis of ubiquitin (MW 8451 Da). They also observed protein loss with long dialysis times (>30 min). In the optimization of drop dialysis, we observed a similar trend:
technical notes ion abundance from peptides (and other contaminants) became lower, with a corresponding increase in glycan ion abundance and MALDI sample spot improvement, as dialysis time was lengthened. This is interesting since glycans observed in plasma with this protocol were significantly lower in MW (
