Data Sheet

AbstractThe Direct Detect™ spectrometer, an infrared (IR)-based spectrometry system working in combination with a novel membrane technology, quantifies amide bonds, which are intrinsic to all proteins and peptides. Application of peptides in solution onto the membrane enables fast drying and rapid and inexpensive quantitation by the Direct Detect™ system, eliminating the need for time-consuming amino acid analysis (AAA).

IntroductionDetermining peptide concentrations accurately and quickly has proven difficult for many researchers. Most commonly used methods for peptide quantitation rely on the weight of the lyophilized powder, absorbance of ultraviolet (UV) light or amino acid analysis. Establishing peptide concentration based on the weight of the lyophilized peptide is inaccurate in most cases, because the analyzed powder can contain a significant quantity (10-70%) of bound water, salts or counterions. Another peptide quantitation method relies on absorbance at 280 nm, and thus can only be used to estimate peptide concentration if tryptophan and tyrosine resides are present in the sequence1,2. Therefore, peptides that do not contain amino acids that absorb light at 280 nm cannot be accurately quantified using this method. While it is possible to determine peptide concentration by measuring absorbance at 205 nm, this measurement is far more sensitive to variations in sample composition, since many solvents and other chemicals will absorb at this wavelength3. A high-quality, dual-beam spectrophotometer is also required to measure UV absorbance in order to reduce the effects of nonspecific absorption and to measure low concentrations. Finally, amino acid analysis, recognized as a gold standard in peptide quantitation, delivers possibly the most accurate peptide quantitation; however, it is expensive and requires time-consuming sample manipulation along with specialized equipment.

The Direct Detect™ spectrometer provides a universal, fast and accurate peptide quantitation method that does not require sample manipulation. IR spectroscopy exploits the fact that molecules absorb specific frequencies characteristic of their structure. To form a peptide, amino acids are covalently linked via amide (peptide) bonds. Amide bonds absorb electromagnetic radiation in multiple regions of the IR spectrum, including the strong band at 1600-1690 cm-1 (“Amide I”; Figure 1). In order to determine protein and peptide concentration, the Direct Detect™ spectrometer uses the intensity of the Amide I band, which is assigned to C=O stretching vibration of the peptide bond (about 80%) with a minor contribution from C-N stretching vibration (about 20%)4.

Application Note

Fast and accurate peptide quantitation using the Direct Detect™ spectrometer

Figure 1. Vibrations responsible for the Amide I band in the infrared spectra of peptides and proteins.

1800 1700 1600 1500 1400.00

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Amide IVibration

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Amide I

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we compared the NIST BSA-based standard curve generated in PBS to curves generated in water, 50% PBS and 50% DMSO. Figure 2 shows a high degree of overlap between the protein-based curve and peptide-based curves, indicating that protein-based calibration curves can be used for peptide concentration determination. Contribution of the solvent to the strength of amide signal seems to be minimal (as evidenced by overlap between curves generated in different solvents). However caution should be exercised during sample preparation when using DMSO and other hydrophobic solvents, since such solvents may spread beyond the sample position on the assay-free card. To obtain best results, the card spotted with peptide solution in a hydrophobic solvent, like DMSO, should be left on the spotting tray until the membrane is white again.

Choosing the right analysis method for peptide quantitationThe instrument is equipped with three analysis methods accounting for variations in sample conditions and spectral features in the analysis region. Analysis method 1 has been developed for proteins and peptides analyzed in a non-interfering environment. Method 1 should be applied to measurements delivering a spectral profile similar to that shown in Figure 1, in which spectra of sample components do not interfere with the Amide I band and the Amide I band does not extend beyond the boundaries of the analysis region (1702-1602 cm-1). The quantitation of peptides displaying wide or multipart Amide I signals should be performed using analysis method 3. This method also permits quantitation of peptides with unusual spectral distributions in the

Materials and MethodsAll IR-based concentration estimations were performed using Direct Detect™ assay-free sample cards and the Direct Detect™ spectrometer. Each card contains hydrophilic spots surrounded by a hydrophobic ring to retain the analyzed sample within the IR beam for convenient sample application and analysis. All measurements were performed using 2 µL of sample solution.

Sample concentration was determined in reference to a calibration method. The Direct Detect™ spectrometer requires a one-time standard curve generation. For reference measurements, the system was calibrated using National Institute of Standards & Technology (NIST) bovine serum albumin (BSA) SRM927d in phosphate-buffered saline (PBS). A series of concentrations covering the instrument dynamic range (0.25 mg/mL to 5 mg/mL) was used to generate the calibration curve. Additional calibration was performed using two commercially available fragments of adrenocorticotropic hormone (ACTH): ACTH 4-10 (Sigma-Aldrich, Catalogue No. A0401) and ACTH 18-39 (Sigma-Aldrich, Catalogue No. A0673). Peptides were dissolved in water and their initial concentrations were estimated by AAA. For peptide-based standard curve generation, stock solutions of the peptides were diluted into water, PBS or dimethylsulfoxide (DMSO).

Performance of the Direct Detect™ spectrometer was assessed using crude peptides, material desalted using C18 cartridges and HPLC-purified stock solutions. To address compatibility with solvents commonly used in peptide studies, we determined concentrations of peptides dissolved in various solvents, including water, DMSO, acetonitrile and methanol.

ResultsAnalysis of infrared spectra collected for an amino acid, a dipeptide and a tripeptide showed that the tripeptide delivered a spectral profile consistent with the Amide I and II distribution observed for proteins, suggesting that the system was capable of analyzing sequences as short as a tripeptide.

Effect of calibration curve type on peptide quantitationThe Direct Detect™ spectrometer is pre-loaded with a standard curve prepared with NIST BSA. We tested the hypothesis that instrument calibration using a protein remained valid for peptide analysis by comparing the preloaded standard curve to the curves generated using a fragment of ACTH (ACTH 4-10). Furthermore,

Figure 2. Overlay of calibration curves generated using NIST BSA and ACTH 4-10. Data points represent the average of 3 replicate samples; measurements were performed by two distinct users to control for potential variability in spotting from user to user.

5.00 6.003.00 4.001.000.00 2.000.00

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NIST BSAACTH 4-10 in 50% PBSACTH 4-10 in WaterACTH 4-10 in 50% DMSO

Amid

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analysis region. For example, some peptides display additional bands (around 1615 cm-1) between the Amide I and II signals. Recent literature suggests that this observed peak may indicate peptide aggregation5, which, if confirmed, would add value to peptide quantitation by the Direct Detect™ spectrometer.

IR-based measurement of peptide concentration is consistent with amino acid analysisThe Direct Detect™ spectrometer has been optimized for use with protein and peptide samples within the concentration range of 0.25 mg/mL - 5 mg/mL. We compared estimated peptide concentrations using protein calibration (NIST BSA), peptide calibration (ACTH 4-10 and ACTH 18-39) and the current gold standard method (AAA). As shown in Figure 3, AAA yielded concentration estimates of 2.88 ± 0.04 mg/mL (peptide A) and 3.67 ± 0.123 mg/mL (peptide B). The concentration determined by using the Direct Detect™ spectrometer for peptide A was 2.814 ± 0.042 mg/mL (against NIST BSA calibration), 2.882 ± 0.007 mg/mL (against ACTH 4-10 calibration) and 3.048 ± 0.114 mg/mL (against ACTH 18-39 calibration). The Direct Detect™ spectrometer estimated the concentration of peptide B at 3.681 ± 0.013 mg/mL (against NIST BSA calibration), 3.659 ± 0.043 mg/mL (against ACTH 4-10 calibration) and 3.395 ± 0.055 mg/mL (against ACTH 18-39 calibration).

Peptide-based calibration enables concentration determination that is highly accurate in the middle of the instrument’s dynamic range but shows increasing error at either extreme of this range (data not shown). In contrast, the preloaded NIST BSA-based calibration curve enables accurate concentration estimations along the entire dynamic range.

Effect of various solvents on peptide concentration estimationPreparation of research grade peptides includes synthesis, HPLC purification and lyophilization. Stock solutions of peptides, predominantly in water or DMSO, are commonly prepared based on the weight of the lyophilized powder. We assessed the compatibility of the Direct Detect™ IR-based system with solvents used in peptide synthesis and research. We analyzed six HPLC-purified and lyophilized peptides of varying lengths, ranging from heptamer to 22-mer. Two of the analyzed peptides were biotinylated and one contained a photosensitive group. Figure 4 shows that the Direct Detect™ spectrometer delivered accurate estimates of concentration, regardless of the solvent used to prepare the peptide solution. All peptides have been HPLC purified, lyophilized and analyzed on C-18 RP to be >98% pure by peak area integration. Weight-based

Figure 3. Two peptides were quantified using the Direct Detect™ spectrometer with both protein- and peptide-based calibration and compared to the reference method (AAA).

Figure 4. The Direct Detect™ spectrometer quantitates peptides dissolved in various solvents.

concentration of each analyzed peptide have been estimated for the double-lyophilized powder.

Analysis of crude and “desalted” peptidesThe most commonly used method for peptide quantitation relies on the weight of the lyophilized powder. Establishing the actual peptide concentration based on the weight of the lyophilized material, especially in crude mixtures, is inaccurate in most cases because the analyzed powder can contain a significant quantity (10-70%) of bound water, salts or counterions. Compared to weighing, use of the Direct Detect™ spectrometer increases the accuracy of peptide concentration estimation, because the IR-based technology quantitates only peptide bonds (not water or salts) present in the analyzed solution. Figure 5 shows that weight-based concentration determination quite often overestimates actual peptide concentration. Material used for generation of the data shown in Figure 5A was cleaved from the resin, precipitated with ether, re-suspended in water and lyophilized. Each peptide was analyzed by HPLC (purity >70%) and mass spectrometry (MS). Results shown in Figure 5B were generated for peptides “desalted” using C18 cartridges. Again, peptide integrity and relative purity (>80%) were assessed by HPLC and MS. It is clear that weight-based concentration determination can be highly inaccurate due to high content of additives present in the powder.

Peptide A

AAANIST BSA

ACTH 4-10ACTH 18-39

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50%DMSO

35% AcCN0.5% TFA

50% MeOH1% AcOH

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3.0

4.03.5

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Conclusion The Direct Detect™ spectrometer offers a new method for peptide quantification, providing an analysis that requires a minimal amount of analyte and delivers results within minutes. Requiring minimal sample preparation and compatible with a wide range of sample compositions, the system makes peptide quantitation both easy and versatile. System accuracy and precision are comparable with results obtained by amino acid analysis, making the Direct Detect™ spectrometer an elegant and accessible peptide quantitation tool. The Direct Detect™ system provides every laboratory the opportunity to incorporate accurate peptide quantitation into everyday experiments. This novel instrument, therefore, has the potential to increase the reproducibility and statistical significance of downstream peptide-based analyses, leading to more productive research.

Figure 5. Weight-based peptide concentration estimation versus Direct Detect™ spectrometer readings. Given that peptide concentrations obtained using the Direct Detect™ spectrometer can be verified by amino acid analysis, these data indicate that weight-based peptide quantitation may overestimate peptide concentrations in many samples. Weight based concentration of each analyzed peptide have been estimated for the lyophilized powder. A. Crude peptides showing >70% purity (by peak area integration; C18 RP HPLC); B. Peptides desalted on C18 cartridges showing >80% purity (by peak area integration; C18 RP HPLC).

Description Qty/Pk Catalogue No.

Direct Detect™ Spectrometer and Starter Kit 1 DDHW00010-WW

Includes:

Direct Detect™ Spectrometer 1

Universal Power Adapter 1

Dell Latitude™ 2120 Netbook and Power Adapter 1

Direct Detect™ Software 1

Netbook Stand 1

Spotting Tray 1

Ethernet Cable 1

Direct Detect™ Assay-free Cards (50/pk) 1

Direct Detect™ Assay-free Cards (50/pk) 1 DDAC00010-GR

Direct Detect™ Assay-free Cards (50/pk) 4 DDAC00010-4P

Direct Detect™ Assay-free Cards (50/pk) 8 DDAC00010-8P

References 1. Edelhoch, H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 1967,

6: 1948-1954.2. Aitken A. and Learmonth M. Protein determination by UV absorption. The protein protocols handbook,

Humana Press 1996: 3-6.3. Scopes, R.K. Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 1974, 59: 277-282.4. Jackson M. and Mantsch H.H. The use and misuse of FTIR spectroscopy in the determination of protein

structure. Critical Reviews in 5. Biochemistry and Molecular Biology 1995, 30(2): 95-120.5. Kupser P. et. al. Amide I and II Vibrations of the Cyclic β-Sheet Model Peptide Gramicidin S in the Gas Phase.

J. Am. Chem. Soc. 2010, 132: 2085–2093.

Pept1

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EMD Millipore, the M logo, and Direct Detect are trademarks of Merck KGaA, Darmstadt, Germany. All other trademarks are the property of their respective owners.Lit No. AN5112EN00, Rev. A BS-GEN-12-06926 8/2012 Printed in the USA. © 2012 EMD Millipore Corporation, Billerica, MA USA. All rights reserved.

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