Analysis of Peptides via Capillary HPLC and Fraction Collection Directly onto a MALDI Plate for Off-line Analysis by MALDI-TOF Application Note 219 Joan Stevens, PhD; Luke Roenneburg; Kevin Fawcett (Gilson, Inc.) Introduction Off-line coupling of capillary HPLC to mass spectrometry has significant advantages to current on-line methodology. Both the HPLC and MS parameters can be individually optimized and off-line coupling provides a means of capturing the HPLC run in time. All proteins and peptides that are separated through the capillary HPLC system are captured on a MALDI target and are analyzed through the MALDI-TOF MS at a convenient time. MALDI-TOF MS provides a means of freezing the sample in time, which allows re-analyzing of the collected fractions from a chromatographed sample as many times as are needed. Hence, fraction collecting the entire sample to a MALDI plate ensures that all information from the chromatographic analysis is retained (i.e., nothing is lost). The MALDILC System described below provides a solution to both micro and capillary HPLC. The detector output from the HPLC is coupled with on-line matrix addition through a micro Tee for direct fraction collection onto a Bruker MALDI plate. The column eluent, after passing through the detector, is mixed with matrix in the micro Tee and deposited onto the MALDI plate. The flow rate of the matrix addition is user-selectable, allowing for optimization of the sample/matrix ratio. November 2003 Page 1 of 8 319319-02
Matrix-Assisted Laser Desorption Ionization (MALDI) MALDI is a method that allows for the vaporization and ionization of non-volatile biological samples from a solid-state phase directly into the gas phase. The sample (analyte) is suspended or dissolved in a matrix (usually in a 1000x molar excess). Matrices are small organic compounds that are co-crystallized with the analyte. The presence seems to spare the analyte from degradation, resulting in the detection of intact molecules as large as 1 million Da. A laser beam serves as the desorption and ionization source. The matrix absorbs the laser light energy, causing part of the illuminated substrate to vaporize. The matrix plume carries the analyte into the vacuum, which aids the sample ionization process. The matrix molecules absorb most of the incident laser energy, minimizing sample damage and ion fragmentation. Once the sample molecules are vaporized and ionized, they are transferred electrostatically into a time-of-flight mass spectrometer (TOF-MS) where they are separated from the matrix ions. The molecules are then individually detected, based on their mass-to-charge (m/z) ratios, and analyzed. Materials & Methods Instruments and Accessories Gilson 350 Micro Pumps for binary gradient Nano Mixer, equipped with: 360-µm OD tubing Gilson 235 Autoinjector, equipped with: 100-µL syringe and rack for 12 x 32-mm vials Gilson 155 UV/VIS Dual-wavelength Detector, equipped with: capillary 35 nl, 8-mm flow cell Gilson 223 Fraction Collector, equipped with: 56-mm arm, spring-loaded probe (1/32 OD x 0.0035 ID x 90 µm PEEK) with Upchurch MicroTee, and rack holder for Bruker AnchorChip 384-well MALDI plate Gilson 402 Syringe Pump, equipped with: 250-µL syringe and matrix valve with matrix/wash solution Discovery BioWidePore C-18 capillary column, 15 cm x 0.32 mm x 5 µ UniPoint System Software version 5.1, 506C Interface, Pentium 4, >2 GHz, 512 MB RAM, 80 GB Hard drive Bruker MS MALDI-TOF XACO, 4.04 acquisition software, Xmass 5.1 data processing software and AutoXecute 5.0 Description of the Procedure: Application of the Sample/Matrix to the MALDI Plate A stock solution of α-cyano-4-hydroxycinnamic acid (matrix) was prepared by dissolving 5.0 mg/ml in 60:40 water:acn An external 402 Syringe Pump pumps the matrix solution at a constant flow through the matrix valve into the Upchurch MicroTee (the rate is adjustable) An Upchurch MicroTee was used to mix the eluent from the detector with the matrix solution (at the very top of the spring-loaded probe) The 223 Fraction Collector with spring-loaded probe was used to spot a mixture of the matrix and eluent on the Bruker AnchorChip 384-well plate and allowed to dry Stock solution of peptides was used for the testing of the HPLC and MALDI FC, containing Val-Tyr-Val, methionine enkephalin, leucine enkephalin, angiotensin II (Simga H-2016 HPLC Peptide Standard Mixture) The total concentration of the peptide mixture in water 0.05% TFA was 220 ng/µl November 2003 Page 2 of 8 319319-02
Photo 1: Gilson 223 Fraction Collector Collection of the fractions directly on the MALDI plate is accomplished with a spring-loaded probe on the 223 (1/32 OD x 0.0035 ID x 90 µm capillary PEEK tubing). Photo 2: Collected Fractions on the MALDI Plate Bruker AnchorChip MALDI plate after fraction collection directly onto the plate. Two different fraction volumes were collected onto the plate: 750 nl was spotted for the first 10+ rows, 1.5 µl was spotted for the last 5 rows. The plate was allowed to dry and then was evaluated by a Bruker MALDI-TOF XACO. November 2003 Page 3 of 8 319319-02
Figure 1: Spring-loaded Probe and Mixing Tee The output flow rate from the detector is introduced into the Upchurch mixing tee at the top of the spring-loaded probe. The matrix solution is introduced at a constant flow via a 402 Syringe Pump to the mixing tee from the side. The ratio of mobile phase to matrix solution is an adjustable parameter within the software. The length of tubing is optimized between the detector and the spring-loaded probe to minimize band broadening; there is a 3.0-µL delay from the output of the UV detector to the tip of the probe. Figure 2: Examples of Band Broadening of Peaks Directly Related to Delay Volume The peak width (PW) is doubled through 4 ft. of 100 µm ID tubing with a delay of 40 µl. November 2003 Page 4 of 8 319319-02
Figure 3: Band Broadening of Peaks Through 2 ft. and 16 in. (respectively) of 100-µm ID Tubing The peak overlays show the amount of peak spreading through 1) 2 ft. of 100-µm ID tubing with a delay volume of 5 µl, and 2) 16 in. of 100-µm ID tubing with a delay volume of 3.0 µl (which is the same tubing delay volume on the MALDI injection collection system presented in this application, hence the peak shape is maintained). Control and Data Handling The MALDILC System was controlled with UniPoint System Software version 5.1. The collection of fractions directly onto the MALDI plate was defined by the control method in the software. The rate at which the MALDI matrix was dispensed is a user-defined parameter. Fractions were collected by time, and multiple collection windows within a run is an available option. Figure 4: UniPoint Method for the MALDILC System The table represents the method used to accomplish MALDI fraction collection and spotting using the Gilson MALDILC System. November 2003 Page 5 of 8 319319-02
Figure 5: MALDI Tray File The tray represents the layout for the Bruker MALDI plate. Wicking positions are available either on or off the plate. The wicking positions are used as a location for the probe to go to in between collection windows. Results Figure 6: Operations List The Operations List represents the flexibility of the MALDILC System. Rates associated with the matrix can be user defined. Time-per-fraction spot can also be optimized for the samples and can vary within a series of samples. Automatic priming of the matrix pump and cleaning of the matrix valve and pump are available options and are automated through the software. Figure 8: Fractions Collected onto a MALDI Plate Graphical depiction of the positions where fractions were collected onto the MALDI plate. Figure 7: Chromatogram of the MALDI Standards The chromatogram is a 1-μL injection (internal loop) of the 220 ng/µl peptide mix (Val-Try-Val, methionine enkephalin, angiotensin II and leucine enkephalin); approximately 55 ng/µl of each peptide. November 2003 Page 6 of 8 319319-02
Figures 8 10: Resulting Spectra from the Collected Fractions The MS spectra in figures 8 through 10 are from the MALDI plate that was spotted with the collected fractions. A spectrum was not obtained for Val-Try-Val (MH+ = 379) because of background interference within the MS. November 2003 Page 7 of 8 319319-02
Summary The 233 Fraction Collector equipped with spring-loaded probe allows direct spotting of fractions on the Bruker AnchorChip 384-well plate; its smaller footprint offers a solution for fraction collection directly onto MALDI plates at a fraction of the cost of larger MALDI spotters The 350 Micro Pumps offer a splitless flow with a flow rate range from 300 nl/min to 50 µl/min UniPoint System Software offers control over the instruments, while still allowing the flexibility to customize the flow rate, rate of matrix addition, and time collection per spot to accommodate the needs of the researcher Conclusion Gilson s MALDILC System offers researchers a total solution for the analysis of proteins/peptides via capillary HPLC Splitless Flow and direct collection of fractions onto a Bruker AnchorChip 384-well MALDI plate. Implementing direct collection of fractions on a MALDI plate eliminates the requirement of manual and/or automated formatting of the MALDI plate prior to analysis. Using an innovative spring-loaded probe (internal peek tubing) eliminates the possibility of damaging the surface or substrate of the MALDI plate. Gilson s 223 Fraction Collector allows incrementing from spot to spot in as little as 0.03 minutes (1.8 seconds per collection spot); the rapid arm movement (<25 cm/sec) of the 223 Fraction Collector is well suited for fraction collection onto a MALDI plate. The springloaded probe demonstrated a repeatability of ±0.25 mm in the X/Y/Z dimensions. The MALDILC System represents Gilson s commitment to offer a complete solution for researchers at a fraction of the investment of other comparable systems. Gilson, Inc. World Headquarters 3000 W. Beltline Hwy., P.O. Box 620027, Middleton, WI 53562-0027 USA Telephone: 800-445-7661 or 608-836-1551 Fax: 608-831-4451 www.gilson.com Gilson S.A.S. 19, avenue des Entrepreneurs, BP 145, F-95400 VILLIERS LE BEL France sales@gilson.com, service@gilson.com, training@gilson.com November 2003 Page 8 of 8 319319-02