mono capillary LCMS Proteomics

mono capillary LCMS Proteomics - Application Note 522...

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Unformatted text preview: Application Note 522 Monolithic Capillary Columns in LC-MS Proteomics INTRODUCTION Polymeric monolithic stationary phases offer an alternative to the classical microparticulate sorbents, bringing important advantages to sample analysis. In contrast to the traditional stationary phases that consist of packed particles, the monolithic separation medium is made of a continuous, rigid polymeric rod with a porous structure. The lack of intraparticular void volume improves mass transfer and separation efficiency, which allows for very fast separations of biopolymers. INSTRUMENTATION All experiments were performed on the UltiMate™ Plus Nano and Capillary LC System equipped with a special 3-nL UV flow cell, and the FAMOS™ Micro Autosampler. The Monolithic capillary column, 200-µm i.d. × 5 cm, made of PS-DVB (polystyrenedivinylbenzene polymer), was thermostatted at 60 °C, using the UltiMate column oven. UV detection was performed at 214 nm, and the flow rate was 3 µL/min (gradient mode). For LC-MS, the system was coupled on-line to a fast-scanning, ion-trap MS (esquireHCT, Bruker-Daltonics) equipped with a nanospray source. Figure 1. Monolithic capillary column in protective housing, 200-µm i.d. × 5 cm. HIGH-RESOLUTION PEPTIDE SEPARATIONS Figure 2 shows the separation of a test mixture consisting of 9 peptides (see Table 1). A gradient from 0–25% acetonitrile in acidified water (0.05% TFA) is performed in 7 min, resulting in a fast baseline separation of all peptides. Peak widths at half height (PWHH) of only 1.6–3.5 s illustrate the fast separations that are achievable using a Monolithic capillary column. TABLE 1. PEAK WIDTH AT HALF HEIGHT (PWHH) FOR PEPTIDES SEPARATED ON A MONOLITHIC CAPILLARY COLUMN Peptide 1. Bradykinin fragment 1–5 2. Vasopressin [Arg ] 3. Methionin enkephalin 4. Leucin enkephalin 8 Retention time min 3.3 3.8 4.0 4.4 4.6 4.9 5.1 6.3 6.4 PWHH s 3.5 1.6 1.9 2.3 1.6 2.5 1.9 2.0 2.6 Peaks: 1. 2. 3. 4. 5. 6. 7. 8 9. Bradykinin fragments 1–5, Vasopressin [Arg8] Methionine enkephalin Leucine enkephalin Oxytocin Bradykinin LHRH Bombesin Substance P 5. Oxytocin 6. Bradykinin 7. LHRH 8. Bombesin 9. Substance P 6 Injected amount: 500 fmol each Detection: UV, 214 nm response time 0.1 s 2 3 4 5 6 7 FAST LC/MS ANALYSIS Figure 3 shows the fast separation of the tryptic peptides from 13 proteins. Peak capacities of up to 150 peaks in less than 15 min are routinely observed. PWHH is in the range of only 3 s, illustrating the tremendous separation performance of Monolithic capillary columns. mAU 8 1 9 2.5 0 0 2 Minutes 4 6 PWHH just 3 s 20432 Intensity 7 × 10 Figure 2. Separation of peptide test mixture. 0.0 0 2 4 6 8 Minutes 10 12 14 20433 Figure 3. Capillary LC-MS separation of 13 digested proteins. Up to 150 peptides separated in less than 15 min (courtesy Dr. Detlev Suckau, Bruker-Daltonik, Bremen, Germany). 2 Monolithic Capillary Columns in LC-MS Proteomics From an arbitrarily chosen peak eluting at 8.6 min and marked with an asterisk (*), a full MS scan was performed (Figure 4). The MS scan revealed this peak consisted of 4 precursor ions. With a scan speed of 26,000 amu/s and unit resolution by the esquireHCT, it was still possible to perform MS-MS experiments of each precursor ion and identify the corresponding peptide. Overall, more than 184 peptides from this 15 min chromatographic run could be identified by MS-MS (Figure 5). 700.01 1 Intensity 5 × 10 357.66 550.87 229.57 A 783.20 985.25 1181.15 869.39 2438.25 0 760.51 3 B Intensity 5 × 10 333.00 873.48 604.51 1080.48 8 D 878.55 0 1067.41 Intensity 7 × 10 C A 721.85 586.06 B 3 615.88 C 1055.18 0 400 600 800 1000 1200 1412.55 1400 20434 Intensity 5 × 10 375.89 480.06 1230.32 741.17 892.39 1401.48 2712.04 m/z 0 Figure 4. MS data of the peak eluting at 8.6 min (courtesy Dr. Detlev Suckau, Bruker-Daltonik, Bremen, Germany). 4 1399.44 D Intensity 5 × 10 0 357.90 1002.54 1172.49 1285.58 1512.52 2626.85 558.21 659.41 887.41 458.90 736.56 500 1000 1500 m/z 2000 2500 20435 Figure 5. MS-MS scan of the 4 detected precursor ions (courtesy Dr. Detlev Suckau, Bruker-Daltonik, Bremen, Germany). CONCLUSIONS Monolithic capillary columns (polymer-based) show excellent separation performance. The same column can be used for both protein and peptide separations. Using short columns of 5 cm length very fast peptide separations with PWHH of a few seconds and of high sensitivity are achieved. Another advantage of the monolithic structure is the very robust column bed, resulting in zero voiding and superior column lifetime. Coupling these columns to ESI-MS results in very fast and sensitive LC-MS analysis, making these columns ideally suited for high-throughput LC-MS proteomics. UltiMate and FAMOS are trademarks of Dionex Corporation. Dionex Corporation 1228 Titan Way P.O. Box 3603 Sunnyvale, CA 94088-3603 (408) 737-0700 Dionex Corporation Salt Lake City Technical Center 1515 West 2200 South, Suite A Salt Lake City, UT 84119-1484 (801) 972-9292 Dionex U.S. Regional Offices Sunnyvale, CA (408) 737-8522 Westmont, IL (630) 789-3660 Houston, TX Atlanta, GA Marlton, NJ (281) 847-5652 (770) 432-8100 (856) 596-06009 Dionex International Subsidiaries Australia 61 (2) 9420 5233 Austria (01) 616 51 25 Belgium (03) 353 42 94 Canada (905) 844-9650 China (852) 2428 3282 Denmark 36 36 90 90 France 01 39 30 01 10 Germany 06126-991-0 Italy (06) 66 51 50 52 Japan (06) 6885-1213 Korea 82 2 2653 2580 20427The Netherlands (0161) 43 43 03 Switzerland (062) 205 99 66 United Kingdom (01276) 691722 LPN 1597 PDF 5/04 © 2004 Dionex Corporation * Designed, developed, and manufactured under an NSAI registered ISO 9001 Quality System. ...
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