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					Enhanced Tryptic Digestion in under                                                                                                                                                                                                                                              Issa Isaac ; Kim May ;
                                                                                                                                                                                                                                                                                                                             1                                                   2




                                                                                                                                                                                                                                                                                 William K. Russell ;
                                                                                                                                                                                                                                                                                                                                                                     2




20 minutes using AFA Technology
                    ™
                                                                                                                                                                                                                                                                                 Jim Laugharn ; David H. Russell
                                                                                                                                                                                                                                                                                                                                                   1                                                                                   2

                                                                                                                                                                                                                                                                                                                                                                                                                                                                Covaris, Inc. • 14 Gill Street, Unit H, Woburn, Massachusetts 01801-1721 USA
                                                                                                                                                                                                                                                                                                                                                                                                                                                  Tel: +1 781-932-3959 • Fax: +1 781-932-8705 • Email: info@covarisinc.com • Web: www.covarisinc.com



Abstract                                 Methods & Material                                                                                                                                 Technology                                         Results
Considerable progress in the field of
                                         • Single protein experiments: Myoglobin was dissolved in 50 mM ammonium bicarbonate pH ~ 8, then reduced with 10 mM DTT
                                           and later alkylated with 20 mM iodoacetamide (all Sigma). Modified porcine trypsin was purchased from Promega, and prepared                      Advantage                                          AFA-assisted tryptic digest @ 37 ˚C outperforms tryptic digest @ 37 ˚C.                                                               AFA-assisted tryptic digest is 10:1 better                                                               AFA-assisted tryptic digest after 20 minutes matches
                                           accordingly.
proteomics has enabled detailed                                                                                                                                                                Covaris AFA                                                                                                                                                                                                                                                                                    overnight classic tryptic digest
studies of the structure and role of     • A sample and a control were prepared; where 130 μL of 0.76 μM Myoglobin was placed into a glass round-bottom microTUBE with                         Focused
                                           a slit septum. Trypsin was added to start the digestion.                                                                                            Transducer
many proteins. However, the bot-
                                         • 5 protein-mix experiments: α-casein (1.3 µg), cytochrome C (10 µg), BSA (12 µg), ribonuclease A (2 µg) and serotransferrin (2 µg)
tom-up workflow still requires con-        were mixed in a total of 100 µL of 50 mM ammonium bicarbonate pH ~ 8. Of the 5 protein mixture, 62.5 µL was reduced and
siderable time and effort to prepare       alkylated as mentioned above to a final volume of 180 µL.

samples for mass spectral analysis.      • For all samples mentioned above, a control sample was incubated in a 37 ˚C bath, while the sample was placed in a Covaris S220X
                                           AFA-generation instrument (Woburn, MA) and processed with the following settings: PIP = 25, Duty Factor = 10, Cycles/Burst =
Protein tryptic digestion is a crucial     100 and T = 37 ˚C.
step in the bottom-up workflow for       • At pre-set time points, 1μL aliquots were mixed 1:1 with 15 mM α-cyano-4-hydroxycinnammic acid matrix, 1μL was spotted on a                      COVARIS AFA
biomarker discovery. The rate of this      target for analysis with ABI MALDI-ToF 4800 MS.
                                                                                                                                                                                            • Isothermal sample processing due to precise
step continues to be a bottleneck,       • For the 5 protein mix, 10 µL of each of the samples at the previously mentioned time points was loaded onto an 150-µm x 15 cm                      control and targeting of AFA energy
                                           column (Vydac) using an LC-Packings autosampler and pumps (LC Packings, Sunnyvale, CA) equipped with robotic spotting
and imposes considerable limita-                                                                                                                                                            • Reproducible processing from sample to sample,
                                           (ProBot, Dionex, Sunnyvale, CA), as previously reported.                                                                                           day to day, and scientist to scientist
tions on acquiring protein sequence      • The LC gradient used for all samples spanned 2-60% (solvent B) over 70 minutes. The eluent was mixed with the matrix-assisted                                                                                                                                                                                                             FIGURE 2: The AFA-assisted process yields 10:1 more intense peptide peaks than does the classic
                                                                                                                                                                                            • Easy to automate                                                                                                                                                                                                                                                                                FIGURE 3: The AFA-assisted process after 20 minutes yields similar peak intensity as that
or identifying a PTM when using a          laser desorption/ionization (MALDI) matrix (7 mg mL-1 α-cyano-4-hydroxycinnamic acid) and a total of 420 spots were obtained                                                                                                                                                                                                              protocol after 20 minutes.                                                                               incubated overnight @ 37 ˚C. For the one protein sample, the intensity and sequence
                                           per sample.                                                                                                                                                                                         FIGURE 1: Comparison of peak intensities at different time points (1, 2, 5, 10 and 20 minutes) between AFA-assisted tryptic                                                                                                                    coverage after 10 minutes is nearly 99% on-par with that obtained from the control sample
mass spectrometer. The need for a                                                                                                                                                              Sonicator                                       digest @ 37 ˚C and classic tryptic digest @ 37 ˚C. Peak intensities for the AFA-assisted aliquots were more intense than those
                                         • MALDI-MS data was acquired using a model 4800 Proteomics Analyzer (Applied Biosystems, Foster City, CA). The 30 highest                                                                                                                                                                                                                                                                                                                            after incubating overnight.
complete, efficient, reproducible,         intensity peaks per spot were fragmented using collision induced dissociation and resulting tandem MS data were searched
                                                                                                                                                                                               (bath)                                          collected from the 37 ˚C water bath. For the 1 minute time points, the AFA-assisted peaks are 3 times as intense as the control
                                                                                                                                                                                               Unfocused                                       sample. For the other time points; 2 minutes, 5 minutes, 10 minutes and 20 minutes, the intensity could be as much as 10X as
and fast tryptic digest is paramount.      against the ncbi database using ProteinPilot software version 3.0 (Revision 114732) with the Paragon™ algorithm (Applied                            Transducer
                                           Biosystems, Foster City, CA). Peak lists were generated via the ProGroup Algorithm built into the ProteinPilot software.                                                                            those collected from the control sample, especially after 10 and 20 minutes of tryptic digestion (Figure 2).




                                              Overview of Covaris AFA Technology                                                                                                                                                               AFA-assisted tryptic digest improves search engine scores                                          Sample\protein                                           BSA                          Serotransferrin                      Alpha S2 Casein                         Ribonuclease A                         Cytochrome C

Introduction                                  Covaris’ patented AFA technology works by focusing high frequency acoustic energy
                                              waves from a dish-shaped transducer. The precisely focused AFA acoustic energy                                                  FIGURE 1
                                                                                                                                                                                                                                               TABLE 1: The data acquired from the digestion using the 5 protein mix further proves the
                                                                                                                                                                                                                                               effectiveness of AFA in enhancing tryptic digestion. Table 1 demonstrates the percent
                                                                                                                                                                                                                                                                                                                                                  Overnight @37 ˚C
                                                                                                                                                                                                                                                                                                                                                  AFA 20 min
                                                                                                                                                                                                                                                                                                                                                                                                           86.7
                                                                                                                                                                                                                                                                                                                                                                                                           91.3
                                                                                                                                                                                                                                                                                                                                                                                                                                                85.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                86.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       61.2
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       66.7
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             83.3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             96.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   71.4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   90.5
                                              waves converge into a small localized area, creating intense mixing (Figure 1).                                                                                                                  improvement in coverage when AFA is used to process the tryptic digestion. We observed
Several accelerated trypsin digest
                                                                                                                                                                                                                                               between 5-26% peptide map coverage improvement for 3 of the 5 proteins in the mixture.             Improvement                                              5%                                                                          8%                                    15%                                   26%
techniques such as digestion under            The Covaris acoustic transducer operates at 500kHz with a wavelength of ~1mm,
                                              which enables acoustic energy to be focused directly on samples in glass tubes and                                                            BATH SONICATORS
extreme pressure, sonication, and             vials in a non-contact and isothermal mode. The Covaris AFA closed-vessel process                                                             • Lack of thermal control
use of an organic-aqueous solvent             prevents sample contamination and heat-induced sample degradation. Common                                                                                                                        The peptide map coverage after treatment with AFA-                                                      The peptide map coverage after treatment with AFA-                                                  The peptide map coverage after treatment with AFA-assisted tryptic digest
                                              sonicators, which operated at lower frequency (around 20kHz) and a wavelength of                                                              • Up to 150zx more energy (heat) applied to the
                                                                                                                                                                                              experiment
                                                                                                                                                                                                                                               assisted tryptic digest matches with that after 5 hour                                                  assisted tryptic digest matches with that after 5 hour                                              matches with that after 5 hour digestion for Cytochrome C
system have been suggested.                   ~100mm, cannot provide the same level of energy control (Figure 2). Sonication                                                                                                                   digestion for BSA                                                                                       digestion for serotransferrin
Unfortunately all of these techniques         technology does not achieve the efficiency of AFA, because it naturally scatters the                                                          • Inconsistent sample processing
                                              acoustic energy, while also generating sample-damaging heat.
have severe limitations such as                                                                                                                                                             • Low throughput processing
                                              With the unique performance characteristics of Covaris AFA, the researcher will
thermal degradation of sample,                achieve unsurpassed reproducibility, control and quality results from their sample
carry-over and cross-contamination            preparation process.
                                                                                                                                                                                               1/8” Probe
risk, and the inability to automate              FIGURE 2             infrasonic      sonic                  ultrasonic                                                                        Sonicator
                                                                                              Sonicator   Diagnostic Imaging                  Sonicator                Diagnostic Imaging
the process. In this poster we will
                                                                                                                                     Sonic                              Ultrasonic
introduce the use of the Covaris                                                     Human
                                                                        1 Hz          1 kHz                    1 MHz
                                                                                                                                                                                                                                                                                                                                                                                                                                                           FIGURE 6: Comparison of peptide map coverage for cytochrome C after tryptic digestion with three different treatments show
                                                                                                                                     1m         100mm        10mm      1mm     100um 10um
Adaptive Focused Acoustics™ (AFA)                                                  FREQUENCY (CYCLES/SECOND)                                          WAVELENGTH (IN WATER)                                                                                                                                                                                                                                                                                similar red and yellow coverage regions for the protein digestion for 6 hours and 20 minutes using AFA treatment. Both improve
                                                                                                                                                                                                                                                                                                                                                                                                                                                           on the peptide map coverage for the cytochrome C tryptically digested at 37 ˚C for 20 minutes.
closed vessel, isothermal, and highly
                                           TRANSDUCER TECHNOLOGY Non-contact, high energy density
automatable technology for use in
accelerating trypsin digest. The                                                                                                                                                            PROBE SONICATOR
                                                                                                                                                                                                                                                                                                                                                                                                                                                             Summary
                                                                                                                                                                                                                                                                                                                                                                                                                                                             Covaris AFA technology has been widely utilized in sample preparation for Next Generation Sequencing (NGS)
methodology demonstrated in this                                                                                                                                                            • Up to 150zx more energy (heat) applied to the
                                                                                                                                                                                              experiment                                                                                                                                                                                                                                                     technology platforms due to its highly reproducible, controlled, isothermal, and closed-vessel sample processing
poster will illustrate not only a                                                                                                                                                                                                              FIGURE 4: Comparison of peptide map coverage for BSA after tryptic digestion with three                                                                                                                       that has been designed with high throughput automated processing capability in mind. As demonstrated, these
                                                                                         • Circular transducer                               • Linear Transducer                            • Lack of thermal control                          different treatments show similar red and yellow coverage regions for the protein digestion                                                                                                                   qualities can be utilized successfully in the proteomics workflow especially with the bottom-up proteomics. Data
significant reduction in digestion                                                       • Serial processing                                 • Parallel processing
                                                                                                                                                                                            • Inconsistent sample processing                   for 6 hours and 20 minutes using AFA treatment. Both improve on the peptide map coverage
                                                                                          FOCAL ZONE
                                                                                                                                                          FOCAL ZONE                                                                                                                                                                                                                                                                                         presented here clearly demonstrates the positive effect AFA has on this one basic element of bottom-up
time, but also a significant increase                                                                                                                                                                                                          for the BSA tryptically digested at 37 ˚C for 20 minutes.                                               FIGURE 5: Comparison of peptide map coverage for serotransferrin after tryptic digestion
                                                                                                                                                                                            • Cross contamination risk                                                                                                                                                                                                                                       workflow. With Covaris AFA, tryptic digest accelerated to 20 minutes from overnight and it does not show any
                                                                                                                                                                                                                                                                                                                                                       with three different treatments show similar red and yellow coverage regions for the protein
in the quality and quantity of data                                                                                                                                                                                                                                                                                                                    digestion for 6 hours and 20 minutes using AFA treatment. Both improve on the peptide map             adverse effects on the quality of the resulting peptides. Better yet, the search engine results demonstrate that
                                                                                                                                                                                            • Low throughput processing
during mass spectrometer analysis.                                                                                                                                                                                                                                                                                                                     coverage for the serotransferrin tryptically digested at 37 ˚C for 20 minutes.                        protein scores and the peptide map coverage are enhanced for the samples processed using AFA vs. the samples
                                                                                                                                                                                                                                                                                                                                                                                                                                                             digested using the classical incubation at 37 ˚C.
                                                                                                                                                                                                                                                                                                                                                        Covaris Inc., Woburn, MA; 2TAMU, College Station, TX
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posted:2/19/2013
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