Protein and peptide separations,

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1 A Quarterly Technical Newsletter Spring Grace Vydac: Leading the Way in Separations for Proteomics Protein and peptide separations, always important to protein chemists and enzymologists, have recently entered the mainstream of molecular biology. In the pre-genomic era, a typical separation targeted a protein with known functional activity - in some cases enzymatic, in others specific binding. The activity served as an assay to track the protein during the separation. Highresolution techniques such as polyacrylamide gel electrophoresis (PAGE), -D PAGE, and reversed-phase HPLC that display a wide variety of proteins or peptides were used primarily for purity analyses and structural studies. With the determination of complete genome sequences, modern advances in mass-spectrometry, and readily available benchtop computing power, the landscape has changed dramatically. High-resolution techniques for protein and peptide separation are now a tool of central importance for proteomics. (Ref. ) Proteomics is the study of the total array of protein products that are coded by open reading frame (ORF) sequences in the DNA of an organism. ORFs have been revealed by complete genome sequencing. The number of genes, estimated from identified ORFs in genomic DNA, varies from about 7,5 for a common bacterium, to approximately Nano/capillary columns for LC/MS/MS SHORTFAST columns for rapid protein isolation, for a fruit fly, and, to, for Homo sapiens. In a typical organism, only a very small fraction of the ORFs can be related to previously known protein products. The vast majority of proteins coded by the DNA, if they are produced at all by the organism, Digest of Crude Extract (Thousands of Peptides) Fragment Mass Spectrum Predicted Fragment Mass Spectrum Search Algorithm Compares Nano/capillary HPLC Separation m/z m/z On-line ES/MS/MS () MS (nd Stage) Match = ORF Assignment Sample (- peptides) () Isolation/ Fragmentation Single Peptide Ion Software Models Peptide Fragmentation have never been isolated and have no known biological function. However, many ORFs are known to coincide with genes that control known phenotypic attributes such as behavior, development, and disease or susceptibility to disease. It is therefore presumed that identification and study of Continued on page ORF minutes MS (st Stage) Peptide Mass Spectrum m/z DNA Grace Vydac is a business unit of W. R. Grace & Co.-Conn.

2 Page Practical Peptide Separations for MS Analysis Driven by the need to increase speed and sensitivity in mass spectrometric analysis of proteins from complex cellular extracts, capillary column chromatography has become the sample pre-separation method of choice complementing, and in some cases supplanting, -D PAGE and -D PAGE. Grace Vydac has introduced a full line of VYDAC high-efficiency nano/capillary HPLC columns to support this trend. Performance of a VYDAC µm i.d. C8 reversed-phase column is demonstrated by the experiment of Figure. In this example, the VYDAC nano/capillary column was used to separate peptides in a tryptic digest of apomyoglobin. One microgram of digest was injected. All chromatograms were developed at a flow rate of µl/min with a gradient from 5% to 7% acetonitrile in.5% formic acid. The column eluate was analysed by direct feed to ESI-MS/MS on a Thermo Finnigan LCQ DECA mass spectrometer. Peptide fragmentation spectra were searched with Thermo Finnigan Turbo-SEQUEST software, resulting in 96.7% coverage of the known myoglobin sequence. The facultative anaerobe Shewanella oneidensis ( is of significant proteomic interest because of its ability to adapt to diverse environments and utilize a wide variety of electron acceptors for energy and growth. The S. oneidensis genome has been sequenced and annotated by TIGR. Using the same VYDAC column, a second experiment was designed to maximize loading capacity and demonstrate the total number of proteins that can be identified from a complex protein mixture. A total of mg of S. oneidensis crude lysate was digested with trypsin (from Promega). Three separate injections of µg were made with the column connected to the mass spectrometer operating in data-dependent MS/MS mode scanning a separate mass range for each injection (m/z -, m/z 98-5, and m/z 8-). Figure shows detailed first-stage MS scans from just a small time segment of the resulting chromatogram Tryptic Digest of Apomyoglobin Relative Intensity (%) VYDAC 8MS 96.7% Sequence ID Time (min) Figure. Total ion chromatogram (TIC) for capillary reversed-phase separation of tryptic digest of apomyoglobin. MS/MS analysis produced 96.7% sequence identification. Tryptic Digest of Shewanella oneidensis Crude Lysate Relative Intensity (%) Relative Intensity (%) Information and data for this article were generously contributed by Nathan C. VerBerkmoes & Robert L. Hettich Organic and Biological Mass Spectrometry Group Chemical Sciences Division Oak Ridge National Laboratory for the m/z - range. Even in this narrow m/z range, the sample is very complex Figure. Examples of LC/MS spectra for analysis of S. oneidensis crude lysate digest. The sample is very complex with many many peptides emerging from the RP column in even the short time segment shown in detail here minutes m/z - Time (min) 5 To maximize the total number of proteins identified, four separate injections of 8 µg each were then made. For each injection, a separate mass range was scanned (m/z - 8, m/z 78-, m/z 8-6, and m/z 58-) in order to increase dynamic range (Ref. ). All data files were searched with the TurboSEQUEST software, and identifications were based on two or more high-scoring peptides per protein. Figure shows the total ion chromatograms from this experiment, which resulted in identification of 8 proteins in minutes. TIC Full scans at second intervals. Even at narrow m/z range, the sample is very complex! Over 6 peptides ID in < minute. 9.7 minutes. minutes m/z m/z m/z 5

3 Page High Sensitivity High Resolution Robust, Long-Life Columns Increased Protein Identification Low or No TFA Requirement Multiple injections made using tryptic digests from S. oneidensis lysates as well as ion exchange fractions totalled over 9 mg. Performance of the VYDAC C8 capillary column validated by injection of the apomyoglobin digest standard showed no significant decline. A final experiment involved coupling two VYDAC µm x 5 mm capillary columns in series for two-dimensional separations similar to the MudPIT methodology described by Washburn, et. al. (Ref. ). The first contained VHP strong cation exchange (SCX) packing, and the second the same 8MS C8 reversed-phase (RP) material as before. Two-dimensional chromatography was performed using a completely automated mobile phase program. Peptides initially adsorbed to the SCX column were eluted progressively by a series of salt bumps into the RP column where they were adsorbed at low organic solvent concentration. Each adsorbed fraction was then eluted from the RP column into ESI- MS/MS by an intervening gradient of increasing organic solvent (acetonitrile) at low salt concentration. The scheme of this experiment is shown in Figure. Preliminary results using this -D LC/MS/MS approach succeeded in identifying 5% more proteins from a single injection than obtained using the -D LC- MS/MS method with multiple injections and multiple mass ranges. Further optimization will involve adjusting the salt bump concentrations to better distribute the MS/MS spectra across the experiment. The preliminary settings resulted in too many spectra from low-salt fractions (Fig. 5), making protein identification difficult, and too few from high salt fractions, resulting in inefficient use of instrument time. A greater number of protein identifications may be possible from single-injection analyses by increasing the number of ion-exchange fractions at the lower salt concentrations (i.e., less than 5%C) with the use of smaller -D LC-MS/MS of Tryptic Digest of Shewanella oneidensis Crude Lysate -8 m/z Time (min) m/z Automated -D LC-MS/MS 78- m/z Time (min) m/z Time (min) 6 Time (min) 6 Figure. Total ion count (TIC) chromatograms produced by scanning four mass ranges in separate -D LC-MS/MS injections. Fragmentation scans resulted in identification of 8 proteins. % C TIC 8 6 Time (min) 75 Time (min) 75 Time (min) 75 Time (min) 75 "Salt Bumps" Solvent Composition A 95% H/5% ACN/.5% Formic Acid B % H/7% ACN/.5% Formic Acid C 5 mm Ammonium Acetate/.5% Formic Acid, ph 5. D 5 mm Ammonium Acetate/.5% Formic Acid, ph 5. Figure. Scheme for -D LC-MS/MS analysis. Salt bumps created from solvent C produce ion-exchange fractions adsorbed to reversed-phase. Solvent D (%) is used to elute the remaining peptides from the ion-exchange column. Peptides on the reversed-phase column are then eluted into ESI-MS/MS by intervening gradients of solvents A and B. MS needle voltage and data acquisition are OFF during salt bumps. salt bumps. In addition, proteins from the high salt fractions can be eluted in fewer discrete fractions, by using larger salt bumps. Additional details of the S. oneidensis experiment are described by VerBerkmoes et al. (Ref. ). References. Spahr et. al., Proteomics (), 9-7 ().. Washburn, M.P., Wolters, D., and Yates, J.R. III, Nature Biotechnology, 9, - 7 ().. VerBerkmoes et. al., J. Proteome Research (In Press). Now available on the web at -D LC-MS/MS Optimization Concerns MS/MS Spectra 8 6 inj. 5%C %C %C %C %C 5%C S. oneidensis Lysate Ion-Exchange Fraction 6%C 7%C 8%C 9%C %D Figure 5. Uneven distribution of MS/MS spectra across salt bump fractions in preliminary -D LC-MS/MS experiment. Adjusting salt concentrations for a more uniform distribution should result in identification of more proteins.

4 Page Proteomics Continued from page normal and abnormal proteins coded by these genes will shed light on how the genes produce their effects and of special interest in pharmaceutical research may generate rational targets for new drug development. The identification of ORF products using mass spectrometry consists of three steps: The extremely complex mixture of peptides produced by enzymatic digestion of a cellular extract is first separated into simpler mixtures. A multistage mass spectrometer then separates and fragments the individual peptides. Fragmention mass spectra are stored on a computer. Finally, sequence analysis software generates model fragmentation spectra expected from known sequences in the ORFs and compares them to the fragmentation spectra found by MS/MS analysis. Unique matching patterns assign peptides to specific ORFs. Once representative peptides have been identified, their presence and concentration can be used to assay expression of ORF products by the organism under various conditions, in various tissues during development, for example and may also be used to guide purification of ORF products from suitable sources. In essence the science of protein analysis and purification has come full circle from using known activities to isolate proteins, to isolating known proteins in an attempt to discover activities. High-performance separations of peptide digests are important because the probability that an MS/MS detector will isolate individual peptides and produce unique fragmentation spectra increases as the number of peptides presented simultaneously to the mass spectrometer decreases. For a complex mixture such as a peptide digest of a cellular extract, an appropriate separation method preceding MS/MS analysis divides the crude digest into a large number of zones or samples, each of which contains a much simpler array of peptides. It is not necessary for individual peptides to be completely resolved prior to MS/MS. It is important, however, that the peptides present in each sample are resolved to simpler mixtures with high recoveries for the first MS stage, thereby permitting subsequent MS stages to produce unique fragmentation patterns that can be unequivocally matched by sequence analysis software. While -D PAGE and -D PAGE separations have been useful, microseparations on capillary HPLC columns more readily lend themselves to reproducible automation and are more easily interfaced to online MS/MS detection. Consequently, high-performance capillary LC/MS/MS has become the method of choice in many proteomics laboratories. Grace Vydac leads the way in separations for proteomics with a unique selection of high-performance nano/capillary HPLC columns containing VYDAC LC/MS-grade wide-pore reversed-phase and strong cationexchange adsorbents for peptide separations. The article on page of this newsletter is an example an application of VYDAC capillary HPLC columns in proteomics. In addition, new VYDAC ShortFast columns, described on page 5, increase laboratory throughput by providing faster high-resolution separations and purifications of larger quantities of proteins. This speeds research and development by facilitating isolation and characterization of identified gene products. References. Peng, J., ad Gygi, S.P., J. Mass Spectrometry, 6, 8-9 (). Upcoming Meetings and Exhibits Come visit Grace Vydac s booth or corporate poster and discuss your nano/capillary, analytical, preparative, and process HPLC column needs with Grace Vydac technical personnel at the following upcoming meetings. ASMS Orlando, FL June -6, CORPORATE POSTER HPLC Montreal, Canada June -7, & POSTER Prep Washington, DC June 6-9, Chinese Peptide Symposium Dalian, China July -6, Bio Expo Japan Tokyo, Japan July -, Protein Society San Diego, CA August 7-, ACS Fall Boston, MA August 9-, Peptide Symposium Korea October AAPS Toronto, Canada November -,

5 Page 5 VYDAC SHORTFAST Columns Speed Protein and Peptide Separations Traditional thinking about column lengths in HPLC has been conditioned by small-molecule separations where partitioning between mobile and stationary phases and theoretical plate counts are important for high resolution separations. This is not the case with larger molecules such as proteins, peptides, and nucleic acids. Adsorption of large, multifunctional molecules is best viewed as all-or-nothing depending on mobile phase conditions organic modifier concentration in reversed phase, and ionic strength in ion exchange. Biological macromolecules are firmly retained below a threshold organic modifier or salt composition characteristic of each specific molecule, and release rapidly to the mobile phase above the threshold. Thus, gradient composition and slope are more important than column length as determinators of resolution in protein and peptide separations. This is good news, because short columns with lower back pressures and lower delay volumes can often be used for rapid protein and peptide separations with high resolution, as demonstrated by the separations in Figures 6 and 7. For proteins and simple peptide mixtures*, VYDAC SHORTFAST columns with cm and 5 cm lengths provide excellent resolution with significant benefits. They contain wide-pore Å reversed-phase and 9 Å ion-exchange packings. They are available in a variety of diameters for a wide range of sample loading requirements. They reduce separation times by to 5 fold, especially important for critical method development applications in combinatorial library screening and synthetic peptide purification. Solvent consumption can be reduced accordingly (up to about %). *Note: SHORTFAST columns are not recommended for separation of complex tryptic digests. Peptides on C8 Reversed-Phase Conventional.6 mm x 5 cm Gradient: 5 to 5% B in min Flow Rate:.5 ml/min Max. Backpressure: 8 psi Conventional.6 mm x 5 cm Gradient: 5 to 5% B in min Flow Rate:.5 ml/min Max. Backpressure: 9 psi Time (min) 8TP C8 Å 5 µm ShortFast.6 mm x 5 cm Gradient: 5 to % B in min Flow Rate: ml/min Max. Backpressure: psi ShortFast.6 mm x 5 cm Gradient: 5 to % B in min Flow Rate: 7 ml/min Max. Backpressure: 5 psi Figure 6. Comparison of peptide separations on Å C8 columns of different lengths. Mobile phase: A =.% TFA (v/v) in water. B =.% TFA (v/v) in ACN. Gradients as shown. Peaks: ) neurotensin -8 (fragment), ) oxytocin, ) neurotensin 8- (fragment), ) eledoisin-related peptide, 5) neurotensin, 6) angiotensin I. High resolution Fast separations High throughput Reduced solvent usage Proteins on C Reversed-Phase Conventional.6 mm x 5 cm Gradient: to 6% B in min Flow Rate:.5 ml/min Max. Backpressure: 8 psi 6 8 Conventional.6 mm x 5 cm Gradient: to 6% B in min Flow Rate:.5 ml/min Max. Backpressure: psi Time (min) TP C Å 5 µm 6 8 ShortFast.6 mm x 5 cm Gradient: to 6% B in min Flow Rate: ml/min Max. Backpressure: 6 psi ShortFast.6 mm x 5 cm Gradient: to 6% B in min Flow Rate: 7 ml/min Max. Backpressure: 5 psi Figure 7. Comparison of protein separations on Å C columns of different lengths. Mobile phase: Same as Figure 6. Peaks: ) ribonuclease A, ) cytochrome c, ) BSA, ) myoglobin.

6 Grace Vydac s nano/capillary columns include four diameters from 75 µm to 5 µm containing six different MS reversed-phase adsorbents and one cation exchanger. This is the world s most comprehensive selection of pre-packed LC/MS columns. SHORTFAST columns also available with other packings. Please consult the Grace Vydac website or price list for a complete listing. To order, contact your local VYDAC distributor or Grace Vydac World Headquarters: Nano/Capillary Columns for LC/MS SHORTFAST Columns Adsorbent Column Size C 5 µm C8 5 µm C8 µm C8 5 µm C8 5 µm (polymeric) (polymeric) (polymeric) (polymeric) (monomeric) 5 µm diphenyl 8 µm SCX 75 µm ID x 5 mm MS MS MS.755 8MS MS MS5.755 VHP µm ID x mm MS5.75 8MS5.75 8MS.75 8MS5.75 8MS5.75 9MS5.75 VHP µm ID x 5 mm MS MS MS.755 8MS MS MS5.755 VHP µm ID x 5 mm MS MS MS.755 8MS MS MS5.755 VHP µm ID x 5 mm MS5.55 8MS5.55 8MS.55 8MS5.55 8MS5.55 9MS5.55 VHP µm ID x mm MS5.5 8MS5.5 8MS.5 8MS5.5 8MS5.5 9MS5.5 VHP8.5 5 µm ID x 5 mm MS5.55 8MS5.55 8MS.55 8MS5.55 8MS5.55 9MS5.55 VHP µm ID x 5 mm MS5.55 8MS5.55 8MS.55 8MS5.55 8MS5.55 9MS5.55 VHP8.55 µm ID x 5 mm MS5.5 8MS5.5 8MS.5 8MS5.5 8MS5.5 9MS5.5 VHP8.5 µm ID x mm MS5. 8MS5. 8MS. 8MS5. 8MS5. 9MS5. VHP8. µm ID x 5 mm MS5.5 8MS5.5 8MS.5 8MS5.5 8MS5.5 9MS5.5 VHP8.5 µm ID x 5 mm MS5.5 8MS5.5 8MS.5 8MS5.5 8MS5.5 9MS5.5 VHP8.5 5 µm ID x 5 mm MS5.55 8MS5.55 8MS.55 8MS5.55 8MS5.55 9MS5.55 VHP µm ID x mm MS5.5 8MS5.5 8MS.5 8MS5.5 8MS5.5 9MS5.5 VHP8.5 5 µm ID x 5 mm MS5.55 8MS5.55 8MS.55 8MS5.55 8MS5.55 9MS5.55 VHP µm ID x 5 mm MS5.55 8MS5.55 8MS.55 8MS5.55 8MS5.55 9MS5.55 VHP8.55. mm ID x 5 mm MS55 8MS55 8MS55 8MS55 9MS55. mm ID x mm MS5 8MS5 8MS5 8MS5 9MS5.6 mm ID x 5 mm MS55 8MS55 8MS55 8MS55 9MS55 VHP85.6 mm ID x mm MS5 8MS5 8MS5 8MS5 9MS5 mm ID x 5 mm MS55 8MS55 8MS55 8MS55 9MS55 mm ID x mm MS5 8MS5 8MS5 8MS5 9MS5 VHP8 mm ID x 5 mm MS55 8MS55 8MS55 8MS55 9MS55 mm ID x mm MS 8MS 8MS 8MS 9MS