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. Author manuscript; available in PMC: 2018 Jan 5.
Published in final edited form as: Methods Mol Biol. 2015;1207:51–62. doi: 10.1007/978-1-4939-1396-1_3

Alkylation of Galectin-1 with Iodoacetamide and Mass Spectrometric Mapping of the Sites of Incorporation

Sean R Stowell, Connie M Arthur, Richard D Cummings, Christa L Feasley
PMCID: PMC5755404  NIHMSID: NIHMS833128  PMID: 25253132

Abstract

Galectins can display unique sensitivity to oxidative changes that result in significant conformational alterations that prevent carbohydrate recognition. While a variety of approaches can be utilized to prevent galectin oxidation, several of these require inclusion of reducing agents that not only prevent galectins from undergoing oxidative inactivation, but can also interfere with normal redox potentials required for fundamental cellular processes. To overcome limitations associated with placing cells in an artificial reducing environment, cysteine residues on galectins can be directly alkylated with iodoacetamide to form a stable thioether adduct that is resistant to further modification. Iodoacetamide alkylated galectin remains stable over prolonged periods of time and retains the carbohydrate binding and biological activities of the native protein. As a result, this approach allows examination of the biological roles of a stabilized form of galectin-1 without introducing the confounding variables that can occur when typical soluble reducing agents are employed.

Keywords: Alkylation, Galectin, Mass spectrometry, Oxidation, Reducing agents

1 Introduction

Unlike most proteins secreted from cells, galectins primarily reside within the cytosol where they can be secreted into the extracellular space through an endoplasmic reticulum and Golgi apparatus-independent pathway [1, 2]. Synthesized on free ribosomes in the cytosol of cells, galectins are maintained in a reduced state prior to secretion from the cell secondary to the reducing environment within the cell [3]. Following secretion, galectins can engage carbohydrate ligands outside the cells [4], which appears to reduce galectin sensitivity to oxidative inactivation in the extracellular environment and allows galectins to participate in a wide range of biological activities, from regulating a variety of cellular activities to directly providing innate immunity [5–10]. However, in the absence of carbohydrate ligand, some members of the galectin family, in particular galectin-1, can undergo intramolecular disulfide oxidation, which results in significant alteration in the protein’s conformation that precludes dimerization and carbohydrate recognition [5, 11–14]. However, in the presence of ligand, galectin-1 experiences enhanced dimerization, which limits formation of monomers that appear to be an intermediate for effective galectin-1 oxidation [5].

While galectin-1 oxidation likely reflects a normal pathway that regulates protein function [15, 16], oxidation can complicate the assessment of galectin-1 activity in the context of other biological systems. As a result, many studies include reducing agents in treatment buffers to reduce the impact of oxidation when assessing the carbohydrate recognition-dependent activity of galectin-1 [17, 18]. While this approach facilitates biochemical assessment of galectin-1 activity, such as carbohydrate binding specificity, when added to biological systems, reducing agents can unfortunately complicate the interpretation of results. For example, while inclusion of reducing agents such as dithiothreitol (DTT) and betamercaptoethanol (βME) can prevent galectin oxidation, these reagents also possess the ability to cross cell membranes where they can induce the unfolded protein response within the endoplasmic reticulum [19–21], likely secondary to preventing appropriate oxidation during protein folding [20]. While utilization of membrane impermeable reducing agents, such as reduced glutathione, can reduce the deleterious intracellular consequences associated with DTT or βME inclusion, these reagents can also reduce proteins normally oxidized on the cell surface, which can likewise alter their biological activity. As a result, a method to prevent galectin oxidation in the absence of residual soluble reducing agents is needed to maintain galectin-1 activity while avoiding the confounding influence of reducing agent inclusion.

Cysteine oxidation often reflects disulfide bond formation, as occurs during galectin-1 oxidation, which can be readily reversed upon inclusion of free thiol reducing agents, such as βME, DTT, or reduced glutathione [22]. As a result, these reducing agents form distinct molecular adducts that do not typically reflect the type of stable interactions commonly observed among other covalent linkages in biological systems [22] (Fig. 1). Thus, reduction of galectin-1 with βME, DTT, or reduced glutathione, followed by elimination of excess reducing agent, results in spontaneous adduct loss as reciprocal intramolecular disulfides form. In contrast, cysteine modification driven by reaction with iodoacetamide or maleimide results in the formation of a thioether, which represents a relatively irreversible adduct that can prevent disulfide bond formation [23] (Fig. 1). Thus, following iodoacetamide alkylation, excess iodoacetamide can be removed from the alkylated protein, allowing galectin-1 to retain a stable adduct that prevents intramolecular disulfide bond formation and inactivation of the protein [23].

Fig. 1.

Fig. 1

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Cysteine residues can be modified with various adducts to reduce intramolecular disulfide bond formation. Reduced glutathione, betamercaptoethanol (βME), and dithiothreitol (DTT) behave in a similar way, forming reversible disulfides with free thiols that can reduce intramolecular disulfide bond formation. In contrast, iodoacetamide (IAM) forms a stable thioether with cysteine that does not readily reverse upon removal of excess iodoacetamide

In this chapter, we describe the process of galectin-1 alkylation with iodoacetamide, including a description of methods required for mapping iodoacetamide incorporation (Fig. 2). In addition, we will discuss approaches for examining the effect of iodoacetamide alkylation on carbohydrate binding activity in addition to methods used to assess the protein’s sensitivity to oxidation (Fig. 3). These methods should be of significant value when seeking to understand the potential role of galectin-1 in a variety of contexts while controlling for protein oxidation.

Fig. 2.

Fig. 2

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Incubation of Gal-1 with iodoacetamide results in acetamide incorporation. (a, b) Mass spectrometry of galectin-1 (Gal-1) (a) or iodoacetamide-treated galectin-1 (iGal-1) (b). (c, d) Mass spectrometry of tryptic fragments of Gal-1 or iGal-1. Peak 2002.47 corresponds to the tryptic peptide fragment from Gal-1, while peak 2116.25 corresponds to same peptide after reaction with iodoacetamide. This research was originally published in the Journal of Biological Chemistry. Stowell SR, Cho M, Feasley CL, Arthur CM, Song X, Colucci JK, Karmakar S, Mehta P, Dias-Baruffi M, McEver RP, Cummings RD. Ligand reduces galectin-1 sensitivity to oxidative inactivation by enhancing dimer formation. 2009 Feb 20;284(8):4989-99 © the American Society for Biochemistry and Molecular Biology

Fig. 3.

Fig. 3

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Alkylation protects Gal-1 from oxidative inactivation. Galectin-1 (Gal-1) or iodoacetamide-treated Gal-1 (iGal-1) was incubated for 24 h in PBS at 37 °C followed by subjection to affinity chromatography over lactosyl-Sepharose. This research was originally published in the Journal of Biological Chemistry. Stowell SR, Cho M, Feasley CL, Arthur CM, Song X, Colucci JK, Karmakar S, Mehta P, Dias-Baruffi M, McEver RP, Cummings RD. Ligand reduces galectin-1 sensitivity to oxidative inactivation by enhancing dimer formation. 2009 Feb 20;284(8):4989-99 © the American Society for Biochemistry and Molecular Biology

2 Materials

2.1 Iodoacetamide Alkylation of Free Sulfhydryls on Galectin-1

  1. Recombinant galectin (see Note 1).

  2. PD10 gel filtration column (GE Healthcare) (see Note 2).

  3. Phosphate Buffered Saline (PBS), standard pH 7.4 (Hyclone).

  4. α-d-(+) Lactose, ACS grade (Fisher).

  5. Iodoacetamide (IAM): 1 M stock freshly made in water (Sigma-Aldrich).

  6. Purified water (dH2O): The use of Barnstead/Millipore water (deionized, UV, and carbon filtered H2O) for all buffer preparations is recommended

2.2 Galectin Activity Assay

  1. Lactosyl-Agarose column (Sigma-Aldrich) (see Note 3).

  2. 2-Mercaptoethanol (2-ME) (Fisher) (see Note 4).

  3. Affinity column loading buffer: Phosphate buffered saline with 2-mercaptoethanol (MEPBS)—0.01 M Na2HPO4, 0.85 % NaCl, pH 7.4 with 14 mM 2-ME.

  4. Affinity column elution buffer: MEPBS with 100 mM lactose.

  5. PD-10 gel filtration column (GE Healthcare) (see Note 2).

2.3 Proteolytic Digestion of Alkylated Galectin-1

  1. Sequencing grade modified trypsin, 1 mg/mL in 50 mM acetic acid, store at −80 °C (Promega).

  2. C18 Sep-Pak: 100 mg cartridge (Waters).

  3. C18 Sep-Pak pre-equilibration solution: HPLC grade methanol, water, 50 % ACN, 0.1 % TFA in water.

2.4 HPLC Separation and Fraction Collection

  1. Vydac analytical C18 column.

  2. HPLC and MS solvents: Purified, deionized H2O, HPLC grade or better is recommended (see Note 5).

  3. Acetonitrile (ACN), LC-MS grade.

  4. Trifluoroacetic acid (TFA), Sequencing grade 99.5 % (Thermo Scientific).

  5. Formic acid, LC-MS grade, 99 % (Thermo Scientific).

    HPLC buffers (see Note 5)

  6. HPLC buffer A: 94.9 % dH2O, 5 % ACN, 0.1 % TFA. Add 50 mL ACN to 1 mL ampoule TFA and dilute to 1 L with dH2O.

  7. HPLC buffer B: 94.9 % ACN, 5 % dH2O, 0.1 % TFA.

2.5 Mass Spectrometry Reagents and Software

  1. Alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution.

  2. MALDI target plate (Bruker).

  3. Methanol: MS grade (Fisher, Optima grade or better).

  4. Acetic acid (Fisher).

  5. Flow splitter (Upchurch).

  6. Peptide standard mixture for external mass calibration: bradykinin, angiotensin I, angiotensin II, ACTH clip 18–39 (Anaspec; Fremont, CA) at 1 nmol/µL in 50 % ACN, 0.1 % formic acid in dH2O.

  7. α-Cyano-4-hydroxycinnamic acid (CHCA) matrix solution, recrystallized (see Note 6): Make fresh daily, 10 mg/mL in 50 % ACN, 0.1 % TFA in dH2O (Sigma-Aldrich).

  8. C18 column (Vydac).

  9. LC-MS buffers: (see Note 5).
    1. LC-MS buffer A: 94.9 % dH2O, 5 % ACN, 0.1 % formic acid.
    2. LC-MS buffer B: 94.9 % ACN, 5 % dH2O, 0.1 % formic acid.

    Mass spectrometry analysis software

  10. GRAMS: Perseptive (see Note 7).

  11. flexAnalysis 2.4, BioTools 3.0: Bruker Daltonics.

  12. ChemStation (Agilent Technology).

  13. BioTools 3.0

2.6 Special Equipment

  1. Centrifugal vacuum concentrator, centrivap (Labconco) (see Note 8).

  2. Fast Protein Liquid Chromatography (FPLC) (see Note 9).

  3. High Performance Liquid Chromatography (HPLC) (see Note 10).

  4. MALDI-TOF(/TOF)-MS: Voyager DE-STR (Perseptive Biosystems) or Ultraflex II (Bruker Daltonics).

  5. ESI-MS: Agilent 1100 MSD ion trap with quaternary pump, diode array detector, and flow splitter into the ion trap (see Note 11).

  6. Syringe pump (Harvard Apparatus).

  7. Fraction collector, such as BioRad 2110.

3 Methods

3.1 Iodoacetamide Alkylation of Free Sulfhydryls on Galectin-1

  1. Frozen galectin stocks should be stored in PBS with 100 mM lactose and 14 mM 2-ME (or other appropriate reducing agent) (see Note 4). Remove stock from storage at −80 °C and thaw on ice.

  2. Equilibrate a PD-10 column with PBS and buffer exchange galectin over equilibrated column into PBS buffer.

  3. Collect 0.5 mL fractions from PD-10 column and determine protein concentration. Pool fractions with protein concentrations greater than 1 mg/mL and add PBS such that the final galectin-1 (Gal-1) concentration is ~2–5 mg/mL in PBS.

  4. To the purified galectin (2–5 mg/mL) sample, add 10 % v/v of 1 M IAM stock and incubate at 4 °C overnight (see Note 12).

  5. Remove excess iodoacetamide by applying the protein solution to a PD-10 column pre-equilibrated with PBS. Elute Gal-1 from PD-10 column with PBS.

  6. Verify Gal-1 activity after iodoacetamide treatment.

3.2 Galectin Activity Assay

  1. Dilute recombinant galectin, both alkylated and non-alkylated, in PBS to the desired concentration (typically 1–20 µM) (see Note 13).

  2. Add 250 µL of galectin PBS solution to a 48 well sterile tissue culture plate.

  3. Incubate this plate at 37 °C in a humidified chamber for preestablished time intervals (see Note 14).

  4. To determine residual activity, remove recombinant galectin solution and apply galectin to 1 mL packed column of lactosyl-agarose that has been equilibrated with PBS (see Note 15).

  5. Once the galectin solution has penetrated the column, add five column volumes of PBS to the column and collect 0.5 mL fractions.

  6. Add 100 mM Lactose in PBS as an elution buffer and continue to collect 0.5 mL fractions.

  7. Determine the approximate protein concentration of each fraction by measuring the OD280 nm. Evaluate the percent of galectin in total lactose eluted fractions as a percent of the total starting material (see Note 16).

3.3 Proteolytic Digestion of Alkylated Galectin-1

  1. To iodoacetamide-treated Galectin-1 samples add sequencing grade trypsin (1 mg/mL stock) at a 1:50 enzyme:substrate (w/w) ratio and incubate digest at 37 °C for 14 h.

  2. To desalt peptides by C18 Sep-Pak, apply digested Galectin-1 peptide sample to a Sep-Pak cartridge pre-equilibrated with 3 mL sequential washes of C18 Sep-Pak pre-equilibration solution.

  3. Then elute unbound species from the Sep-Pak by washing with 3 × 1 mL of C18 Sep-Pak pre-equilibration solution.

  4. Finally, elute peptides with 3 × 1 mL of 50 % ACN, 0.1 % TFA in water.

  5. Dry samples by rotary evaporation to 10–20 µL. Tryptic peptides from this step can be analyzed directly (go to Subheading 3.6 below) or after further purification (continue to Subheading 3.4) by LC-MS(/MS).

3.4 HPLC Separation and Fraction Collection

  1. Add an equal volume (10–20 µL) of HPLC buffer A to Galectin-1 tryptic peptides.

  2. Inject tryptic peptides onto the Vydac analytical C18 column pre-equilibrated with HPLC buffer A.

  3. Elute buffer salts and unbound peptides with a two column volume of 98 % buffer A/2 % buffer B wash at 1 mL/min flow rate.

  4. Separate peptides using a 1 mL/min linear gradient from 2 % buffer B to 100 % HPLC buffer B over 80 min, monitoring UV absorption at 215 nm. Collect 1 min fractions.

  5. Concentrate fractions by vacuum centrifugation to a uniform volume of 100 µL.

3.5 MALDI-TOF and TOF/TOF Analysis

  1. To analyze HPLC separated peptide fractions in positive or negative ion mode, spot 1 µL of each peptide fraction on a ground steel MALDI target plate (Bruker), add 1 µL of α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution, and dry under vacuum.

  2. In parallel, prepare a peptide standard spot with 1 µL of the peptide calibration mixture and 1 µL of CHCA matrix solution.

  3. Analyze galectin peptides with a Ultraflex II MALDI-TOFTOF MS in reflectron positive ion mode with an accelerating voltage of 23 kV. Acquire spectra with 66 Hz laser frequency and sum 1,000–2,000 individual spectra for each sample.

  4. Externally calibrate the TOF with the peptide calibration mixture using flexControl 2.4 software.

  5. Galectin-1 peptides observed in the MS are accelerated by 8.5 kV and selected by a timed ion gate for MS/MS fragmentation.

  6. TOF/TOF fragmentation is achieved by accelerating the fragment ions to 19 kV using the LIFT apparatus.

3.6 ESI-MS, Direct Infusion

  1. Combine peptide fractions with an equal volume of methanol with 1 % acetic acid (100 µL) and directly infuse (5 µL/min) into the Agilent 1100 LC/MSD instrument equipped with an ion trap mass analyzer by a syringe pump.

  2. Trap parameters are set as follows: Dry nitrogen is introduced into the capillary region at a flow rate of 5 L/min and heated to 220 °C. Compound stability is set to 35 % and trap drive level parameters are 70 %. Nebulizer gas pressure is set to 15 psi and the spray voltage is adjusted to 3 kV.

  3. Use helium for collision-induced dissociation.

  4. MS and tandem MS (MS/MS) data were acquired and processed using Bruker Trap software 4.1 in our lab.

3.7 LC-MS Analysis (see Note 17)

  1. Inject galectin-1 tryptic peptides (10–20 µL) on an Agilent 1100 HPLC-MSD-Trap with a 4.6 × 250 mm C18 column pre- equilibrated with LC-MS buffer A.

  2. Use a flow rate of 0.5 ml/min and a linear gradient 2 % LC-MS buffer B to 95 % LC-MS buffer B over 95 min and split the flow 1:10 using a flow splitter and direct 1/10 total column elution into the MSD trap.

  3. Trap parameters should be set the same as in Subheading 3.6 with the following exceptions: Dry nitrogen is introduced into the capillary region at a flow rate of 8 L/min and heated to 300 °C. Compound stability is set to 30 %. Nebulizer gas pressure is set to 25 psi.

  4. MS/MS fragmentation should be data driven to select and fragment the most abundant three ions.

3.8 Data Processing and Analysis

  1. Use flexAnalysis 2.1 software for MALDI-MS spectra processing and peak picking.

  2. Use ChemStation software to process ESI-MSD trap data.

  3. Use BioTools 3.0 software for peptide MS/MS database searching and de novo sequencing.

Acknowledgments

This work was supported in part by grants from the National Blood Foundation, American Society of Hematology and Hemophilia of Georgia to S.R.S.

Footnotes

1

Purification of recombinant galectin is reported elsewhere [5].

2

Alternative gel filtration or desalting columns can be utilized (spin columns, etc.) but this desalting step should be rapid and remove excess lactose used to affinity purify galectin.

3

Buffer exchange protein into buffers containing no lactose before application of cellular extracts or purified proteins.

4

2-Mercaptoethanol or DTT can be utilized as a reducing agent; however, both should be removed from galectin before use in biological assays as both of these thiols also possess the ability to cross cell membranes where they can induce the unfolded protein response within the endoplasmic reticulum [19–21] likely secondary to preventing appropriate oxidation of proteins during protein folding [20]. Thus, each may cause cell damage if not removed prior to biological assays.

5

All FPLC and HPLC buffers should be vacuum filtered with a nylon filter (0.22 µm, 47 mm) and degassed with a helium sparge or vacuum degassed with sonication.

6

Recrystallize CHCA matrix as follows: Mass 5 g CHCA into a clean, dry beaker. Add 10 mL hot methanol and heat the matrix/methanol solution. Add enough methanol (the minimum amount required) to dissolve all of the CHCA. Add 100 mg of activated charcoal and filter the CHCA/methanol solution. Cool the filtrate to room temperature, then cool to 4 °C on ice. Vacuum filter CHCA crystals and wash with ice-cold water. Store at −20 °C in a desiccated container.

7

Data processing and handling on mass spectrometers is instrument and vendor dependent. A variety of software packages are available and may be utilized. Software tools that de novo sequence peptides from MS/MS fragmentation data can also be used for determining sites of iodoacetamide incorporation.

8

We used Labconco centrivap containing a glass lined cold trap chilled to −50 °C with an Edwards vacuum pump capable of reaching 50 mTorr or equivalent.

9

Our lab used AKTA FPLC for protein purification and gel filtration.

10

Use a HPLC system capable of high pressure pumping, flow rates of 0.1–10 ml/min and a detector capable of monitoring at 215 nm or other UV-Vis wavelengths. We used a Beckman System Gold with a UV-Vis detector system equipped with an analytical C18 column (Vydac 4.6 × 250 mm) for protein separations.

11

There are many additional systems available including nanoLCMS versions capable of mass spectrometric peptide mapping. MSn studies can be performed (if needed) by direct infusion of peptide fractions collected from the HPLC.

12

Alkylation of free sulfhydryls can be achieved in shorter incubation times at elevated temperatures. This may be determined empirically.

13

This is commonly done by examining galectin at a similar concentration as employed in biological assays, which can range from 1 to 20 µM. As a control, wells containing 14 mM βME or 3 mM DTT should also be included in separate plates.

14

This can range from examining galectin hours to days following initiation of the incubation period. There are many methodological approaches to inducing protein oxidation. We prefer to measure the potential impact of protein oxidation following protein modification by incubating galectins in a similar environment to which they will be exposed during a typical incubation with cells as this will be the commonly employed experimental situation where galectin oxidation may significantly impact the biological outcome of galectin–ligand interactions, but where inclusion of reducing agents can obfuscate results.

15

It is possible, depending on the protein concentration employed, that oxidative inactivation will result in protein aggregate formation. If this occurs, the final concentration of soluble galectin should be noted and may be used to calculate the overall loss of protein activity secondary to galectin oxidation.

16

If determining galectin protein concentration by measurement of OD 280 then be sure to determine the concentration using the extinction coefficient. Ensure that the column is not saturated such that active galectin is found in the flow through simply because the column limit has been reached. This can be achieved by examining for potential flow through in the reducing agent included control and/or by examining the saturation limit prior to experimental setup.

17

Peptides analyzed by this method are described in an off-line LC-MS manner. There are numerous other high-resolution LC-MS systems that can be substituted for the ones described here.

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