Multidimensional Bioseparations Using -Exchange and Reversed-Phase Monolith Columns Kelly Flook, Andy Woodruff, Charanjit Saini, Srinivasa Rao, Jim ThayeThayerr,, Maria Rey, Yuri Agroskin, and Chris Pohl Dionex Corporation,Corporation, Sunnyvale,Sunnyvale, CA USA

Introduction We will demonstrate here how ProSwift ion-exchange and reversed- phase columns can be used for the multidimensional separation of Historically analysis has relied on 2DE (two-dimensional elec- crude protein samples towards detection and identification by mass trophoresis) to isolate of interest. However, the increased need spectrometry. for speed and sensitivity is pushing the limits of this technology. HPLC alternatives are being developed to overcome the present limitations of protein analysis. The advantages of using HPLC over 2DE are immense: Instruments • No long gel runs ICS-3000 Dual Ion System (Dionex Corp. Sunnyvale, CA) equipped with LPG DP-3000 pump, Detector/Chromatography • No gel removal steps module (DC), ICS-Series Variable Wavelength Detector (VWD), and • No gel interactions AS Autosampler. • Fewer size limitations (small peptides to large proteins) ® • No need for staining with in-line UV detection UltiMate 3000 Chromatography System (Dionex Corp. Sunnyvale, CA) with LPG-3200A pump, TCC-3100 column oven, VWD-3400 variable • Greater sensitivity wavelength detector, and WPS-3000 autosampler. • Ease of automation • Multiplicity of HPLC separations (e.g. ion-exchange to reversed- Software: Chromeleon® 6.8 Chromatography Management Software phase) (Dionex, Sunnyvale, California) was used for system control and data • Direct interfacing to a variety of MS systems processing.

The development of monolithic technology is expanding the boundaries Columns: ProSwift product line, Dionex, (Sunnyvale, California). of HPLC for both small molecule and bioseparations.

Using ProSwift™ monolith columns, the separation of biomolecules can Product Line be achieved at elevated linear velocities with little to no loss of resolu- tion. This range of columns also provides high mass loading capaci- Reversed-Phase Ion-Exchange ties, making them excellent for the first-dimension separation of crude samples. Alongside excellent batch-to-batch reproducibility, the unique 4.6 × 50 mm 1 × 100 mm monolith design and inert column housing provide the excellent pH 4.6 × 50 mm 1 × 50 mm stability required for column sterilization. ProSwift RP-3U In Development ProSwift WAX-1S ProSwift WAX-1S 064298 064294 066642

In proteomic analysis, sample availability is often limited. Improved ProSwift RP-2H ProSwift SAX-1S ProSwift SAX-1S sensitivity, high mass loading capabilities, and a variety of separat- 064296 064293 ing mechanisms are required to analyze low copy number, membrane, ProSwift RP-1S ProSwift WCX-1S ProSwift WCX-1S highly basic or acidic, or posttranslationally modified proteins. 064297 064295 066643 ProSwift SCX-1S Key: Current Current New Polymeric ion-exchange monoliths are designed to maintain good Products Development Products resolution at high loading capacities. Introduction of 1-mm analogues 23874-03 of the current 4.6-mm ion-exchange columns extends to capabilities of the product line by bridging the gap between analytical and micro-bore Figure 1. ProSwift family tree. columns to provide increased sensitivity with lower solvent consump- tion while still allowing the use of standard analytical HPLC systems.

Pittcon 2008 Presentation Pittcon 2008 Presentation 1 ION-EXCHANGE CHROMATOGRAPHY Table 1. Isoelectric Points and Molecular Weights of Studied Proteins Proteins are made up of acidic, basic, and neutral peptides. At a given Protein Name Mwt pI PN Retention pH, depending on the amount of acidic and basic peptides, the protein Albumin, chicken egg 44287 4.54 (4.43-4.66) A2512 SAX will become neutral. This pH is known as the , pI. Trypsin Inhibitor 21000 4.6 T1021 SAX Lactoglobulin A, β 18363 4.83 L7880 SAX • At a pH above the pI, a protein will have a negative net charge and holo-Transferrin 76-81 kDa 5 T4132 SAX

below it, a positive net charge. Lactoglobulin B, β 18726 5.31 L8005 SAX • The ion-exchange stationary phase is charged, either positive or Transferrin 79000 5.5 T3309 SCX negative. Ionized proteins will either be attracted or repelled depend- Albumin, bovine 66430 5.6 A0281 SAX ing on the charge of the protein. Albumin, Human 66478 5.85 A3782 SAX • A cation-exchange column surface is negative and positively charged apo-Transferrin 79000 6 T1428 SAX proteins will be retained. Conalbumin 77776 6.85 C0755 SAX, SCX • An anion-exchange column surface is positive and negatively Crystallin, β 80000 6.9 C5163 SAX, SCX charged proteins will be retained. Chymotrypsinogen A, α 25600 8.97 C4879 SCX • takes place in one of two ways: Ribonuclease A 13700 9.6 R5500 SCX Cytochrome C, Yeast 12588 9.86 C2436 SCX 1. By the addition of counter which are attracted to the surface more strongly than the protein, taking its place on the surface and Cytochrome C, Bovine 12200 10.37 C2037 SCX causing it to elute. Cytochrome C, Equine 12384 10-10.5 C7752 SCX Lysozyme 14700 11.35 L6876 SCX 2. By adjusting the pH of the system so that the protein is no longer ionized and will no longer be retained on the column. Protein pI information was gathered from Sigma Aldrich product info pages. Crude protein samples contain a mixture of proteins with very different Swiss-Prot info was used as an estimation where data was not available from Sigma-Aldrich. Retention mechanism obtained by anion and cation exchange chromatography. pIs. The column and eluent used depend greatly on the mixture and protein of interest in the sample. At pH 6.0, the cation-exchange column will be negatively charged and retain protein with a pI above this pH. The anion-exchange column will be positively charged and will retain proteins with a pI below this pH. If the protein has a pI of 6 it will be neutral and unretained.

• Mechanism of retention: charge-charge interaction Cationic residue/site • Separation based on total charge of protein on the protein • Highly dependent on ionic strength + • Highly dependent on pH – • Elution by increasing ionic strength or pH gradient Column: ProSwift SCX-1S, 4.6 × 50 mm Anion-exchange Flow Rate: 2 mL/min + site covalently Gradient: 0–1M NaCl in 10 mM Sodium phosphate, bound to resin pH 6 in 5 min Inj. Volume: 10 µL Temperature: 30 °C

25076 Detection: UV, 214 nm Sample: 1 mg/mL each (Traces are labeled)

Figure 2. Protein interaction.

β Crystallin mAU Conalbuman Cytochrome C, Bovine Lysozyme Cytochrome C, Yeast α Chymotrypsinogen A Cytochrome C, Equine

Ribonuclease A

Transferrin

0 2 4 6 8 Minutes 25067

Figure 3. Proteins retained at pH 6.0 on strong cation-exchange column.

2 Multidimensional Bioseparations Using Ion-Exchange and Reversed-Phase Monolith Columns Column: ProSwift SCX-1S, 4.6 × 50 mm Gradient: 1% B for 1 min, Eluents: (A) 10 mM Sodium phosphate, 1–100 % B in 5 min Column: ProSwift SAX-1S, 1.0 × 50 mm pH 7.6 Inj. Amount: 20–1000 µg Flow Rate: 0.2 mL/min (B) 1 M NaCl in eluent A Temperature: 30 °C Gradient: 0–1 M NaCl Flow Rate: 0.2 mL/min Detection: UV, 280 nm in 10 mM Sodium phosphate, Sample: Ribonuclease A, 10 mg/mL pH 6 in 12 min Inj. Volume: 5 µL Temperature: 30 °C Detection: UV, 214 nm PWHH (min) Sample: 1 mg/mL each (Traces are labeled) 0.078 1000 µg 0.077 960 µg mAU 0.073 800 µg 0.067 640 µg 0.061 480 µg 0.051 320 µg mAU 0.047 160 µg 0.043 80 µg 0.043 40 µg Trypsin Inhibitor 0.042 20 µg β-Lactoglobulin A β-Lactoglobulin B 0 Minutes 4 25069 HSA BSA β Crystallin Figure 5. Dynamic loading of ProSwift SCX-1S, 4.6 × 50 mm. Ovalbumin Conalbumin holo-Transferrin apo-Transferrin

Peak width at half height 0.326 Column: ProSwift SAX-1S, 1 × 50 mm values in minutes are shown Eluent: (A) 10 mM Tris, pH 7.6 0 4 8 12 16 (B) 1 M NaCl in Eluent A Minutes Gradient: 5–55% B in 13 min, 25068 0.230 hold for 2 min Flow Rate: 0.2 mL/min Inj. Volume: Variable (2–40 µL) Figure 4. Proteins retained at pH 6.0 on strong anion-exchange column. 0.188 Detection: UV, 280 nm Sample: Trypsin inhibitor (10 mg/mL) mAU 400 µg 240 µg High Mass Loading and Fractionation 160 µg 80 µg 0.146 • ProSwift Ion-exchange monolith columns are designed to allow a 40 µg 0.130 high mass loading towards preparative analysis (Figure 5). 20 µg 0.131 • The 1-mm format columns show high loading capacity (Figure 6)

coupled with excellent mass sensitivity (Figure 7), making them 0 4812 160 an excellent choice for analysis where sample is limited or where Minutes a sample contains the protein of interest in low abundance. Large amounts of protein can be loaded without loss of resolution of this 0.35 0.30 protein. 50% increase in PWHH = 158 µg 0.25

• Determination of dynamic loading capacity by 50% increase in (min ) 0.20 PWHH can be deceiving. For a column with excellent resolution, far 0.15

Peak width at half height 0.10 more protein than calculated can be loaded before resolution of the 10 100 1000 Trypsin Inhibitor (µg) protein of interest is lost. 25070

Figure 6. Dynamic Loading of ProSwift SAX-1S, 1 × 50 mm.

Pittcon 2008 Presentation 3 20 Column: WAX-1S, 4.6 × 50 mm Column: ProSwift WAX-1S, 1 × 50 mm Flow Rate: 2.1 mL/min Eluents: (A) 10 mM Tris, pH 7.6 (B) 1 M NaCl in eluent A Gradient: 5–55% B in 13 min mAU Flow Rate: 0.2 mL/min Column: See chromatogram Inj. Volume: 1.3 µL Eluents (A) 10 mM Tris, pH 7.6 Temperature: 30 °C 2 (B) 1 M NaCl in 10 mM Tris, Detection: UV, 280 nm 1 3 pH 7.6 Sample: E. Coli proteins,1.25 mg/mL –0.5 Gradient: 5 to 55% of B in 13 min, 0510 15 hold for 2 min Minutes Flow Rate: See chromatogram mAU Inj. Volume: 1.3 µL Column: WAX-1S, 1 × 50 mm 20 Detection: UV, 280 nm Flow Rate: 0.2 mL/min 2 Protein Mix: 1. Ovalbumin 1 mg/mL 2. Trypsin Inhibitor 1 3. Insulin Chain A 0.25 mAU 3 1

–2 0 5 10 15 0510 15 Minutes Minutes 24269 24270

Figure 7. Sensitivity of 1-mm column vs 4.6-mm column. Figure 8. Separation of E. coli proteins on the ProSwift WAX-1S, 1 × 50 mm.

Fractionation of Crude Protein Column: ProSwift SCX-1S, 4.6 × 50 mm Flow Rate: 2 mL/min 0.105 Samples Gradient: 0–1M NaCl in 1- 0.17 10 mM Sodium phosphate E. coli protein sample (BioRad, Hercules, CA) (at various pH), in 5 min Inj. Volume: 10 µL Temperature: 30 °C AU • About 200 intact proteins can be isolated from E. coli K-12 (strain Detection: UV, 214 nm MG1655), resulting in over 200 modular units. Sample: E. coli proteins, 2.3 mg/mL • A modular unit is a protein element of at least 83 amino acids that 2.5 1- 0.17 0 has an independent biological function. Most E. coli proteins contain -0.011 0.01 0.50 1.00 1.50 2.00 2.50 3.23 one module. Proteins encoded by a gene that has undergone a gene Minutes fusion event contain two or more modules.

• In order to study the structure of each protein fractionation, separa- AU tion and subsequent analysis by MS is required. Eluent pH 2.0 • Separation and isolation by 2DE is labor intensive and unsuitable for 3.0 4.0 instances of low copy number. 6.0 7.6 • Separation using allows the first-dimensional separa- 0 tion by protein pI. –0.5 • The majority of E. coli proteins are anionic at pH 7. Therefore anion- 0 2.0 4.0 6.0 8.0 Minutes exchange chromatography is desirable for the isolation and detection 25071 of the widest range of proteins. • Separation by cation exchange at low pH allows the specific isolation Figure 9. Separation of E. coli by strong cation exchange at various pHs. of the few cationic proteins. E. coli proteins (200 µg) were loaded onto a 1-mm ProSwift SAX-1S column and fractions were collected at 2 minute intervals (Figure 10). Each fraction was then separated using a prototype 1-mm monolithic RP column (Figure 11). This column is housed in inert housing, ensuring stability at high pH to enable sterilization often required for bioapplications. Fraction 1 was then separated by cation exchange at low pH (Figure 12).

4 Multidimensional Bioseparations Using Ion-Exchange and Reversed-Phase Monolith Columns Column: ProSwift SAX-1S, 1.0 × 50 mm 140 Column: ProSwift SCX-1S, 4.6 × 50 mm Flow Rate: 0.2 mL/min Flow Rate: 2 mL/min Gradient: 0–1M NaCl in 10 mM Sodium phosphate, pH 6 in 30 min Gradient: 0–1M NaCl in Inj. Volume: 10 µL 10 mM Sodium phosphate, 10.2 Temperature: 30 °C pH 3 in 5 min Detection: UV, 280 nm Inj. Volume: 10 µL Sample: E. coli proteins, 20 mg/mL Temperature: 30 °C Detection: UV, 214 nm Sample: ProSwift SAX-1S 5,500 mAU E. coli fraction 1

mAU

0 –1.0 0 1.0 2.0 3.0 Minutes

mAU 123456789101112 13 14 15 16

0

–20 0 3 6 9 Minutes 25074

–500 0510 15 20 25 30 35 40 Figure 12. Fraction 1 by Strong Cation Exchange. Fraction 1 contains proteins Minutes 25072 with a pI < 6.

Figure 10. High mass loading of E. coli on ProSwift SAX-1S, 1 × 50 mm. SUMMARY • ProSwift monoliths are a new family of columns developed for 500 Column: ProSwift RP-Prototype, Flow Rate: 0.2 mL/min protein separations. They offer high speed, high resolution, and high 1 × 250 mm Gradient: 0 min 5% A, 85% C, 10% D Eluents: (A) Acetonitrile 1 min 5% A, 85% C, 10% D loading capacity. (B) Not Used 6 min 90% A, 0% C, 10% D (C) Water Hold for 9 min • Monolith technology allows excellent scalability, reproducibility, and (D) 1% TFA in water (v/v) Detection: UV, 214 nm Inj. Volume: 10 µL overall performance. Sample: E. coli SAX fractions • New techniques have been successfully employed to allow prepara- mAU tion of formats >500 nm. • ProSwift (1-mm) columns allow increased sensitivity and bridge the gap between analytical and microbore columns. • ProSwift ion-exchange columns now include SCX-1S, available in 4.6 × 50 mm format, and a SAX-1S in a 1 × 50 mm format. 0 • Due to the high capacity of ProSwift IEX columns, they are ideally suited for the first dimension in a multidimensional chromatography –100 0 5.0 10.0 15.0 17.4 separation. Minutes 25073

Figure 11. Reversed-phase analysis of SAX protein fractions.

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