TN-1076 APPLICATIONS TN-1076 ... Mobile Phase: A: 0.1 % Trifluoroacetic acid in Water B: ... netex...
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APPLICATIONS TN-1076 Page 1 of 4 For additional technical notes, visit www.phenomenex.com High Resolution LC/MS Analysis in Peptide Mapping Using Kinetex ™ HPLC Columns Michael McGinley and Jeff Layne Phenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501 USA The improved efficiency and resolution that Kinetex columns pro- vide is often used for decreasing chromatographic run times; for peptide mapping the improved performance of Kinetex columns can be used to get better resolution of closely eluting peptides. This improved peptide resolution allows one to better identify and quantitate low-level and post-translationally modified peptides. In addition to increased efficiency, Kinetex columns demonstrate good selectivity, porosity, and inertness, all of which have a critical impact on the usefulness of Kinetex columns for peptide mapping applications. Introduction Reduced run time, leading to better throughput, is typically cited as the principle reason for using next generation HPLC columns (UHPLC, monolith, or core-shell). For small molecule separations, bioanalytical separations resolution of closely related peptides as well as improved sensitivity of low abundant components, are the key parameters of interest to most analytical biochemists. Peptide mapping is the principle bioanalytical application where resolution is important; proteins are complex biopolymers that of- ten have post-translational modifications (PTMs) that can affect the activity and immunogenity of a protein therapeutic. Since modifica- tions are only a slight chemical change in the overall protein, pro- teins must be enzymatically fragmented into dozens or hundreds of peptides that are then separated by reversed phase HPLC and positively identified by LC/MS. Good resolution of closely related peptides is critical for identifying PTMs and next generation HPLC columns with higher efficiency have allowed for more accurate information from complex protein therapeutics like Ig-G proteins, which represent a majority of candidates currently in clinical trials. However, high efficiency is not the only important parameter in peptide mapping of complex proteins. HPLC media must have high selectivity of minute chemical differences, have acceptable loading of analytical amounts of digested protein so that low level PTMs can be detected, and media must maintain good performance in MS-compatible buffers used for such analyses. Kinetex, a recently introduced core-shell media, was evaluated against popular fully porous media as well as other superficially porous media in re- gard to peptide mapping performance, peptide loading, and per- formance in volatile buffers and with Ig-G peptide separations to demonstrate the advantages of such a media over existing chro- matographic solutions. Experimental Recombinant and natural proteins and peptides, dithiothreitol (DTT) and iodoacetic acid (IAA) were purchased from Sigma Chemicals (St. Louis, MO, USA). Recombinant human protein Interferon-alpha was obtained from Abcam Inc. (Cambridge, MA, USA)). Solvents were purchased from Fisher Scientific (Fairlawn, NJ, USA). Mobile phase modifiers and Asp-N endoproteinase were obtained from EMD (San Diego, CA). Lys-C endoproteinase was obtained from Wako Chemicals (San Diego, CA). Sequencing grade trypsin was purchased from Roche Applied Science (Indianapolis, IA, USA). Hu- man Ig-G2 was obtained from Dako (Carpentaria, CA). Kinetex 2.6 µm C18 columns (50 or 150 x 4.6 mm) were used for HPLC analysis (Phenomenex, Torrance, CA). HALO ® C18 columns were obtained from Mac Mod (Chadds Ford, PA) and XBridge ™ C18 columns were purchased from Waters ® (Milford, MA). For BSA digests, protein was digested with sequencing grade trypsin 1:50 E/S in 0.1 M NH 4 HCO 3 pH 8.0 at 37 ˚C for 18 hours then quenched with TFA or Formic acid before injection. Differ- ent gradients of TFA/Water or Formic acid/Water from 5 to 45 % B (Acetonitrile) over 30 minutes were used based on the appli- cation shown. For peptide mixtures, fast gradients from 5 to 50 % in 5 minutes were used. HPLC analysis were performed on an Agilent 1100 HPLC equipped with an autosampler, column oven, and MWD using ChemStation software (Agilent, Santa Clara, CA). Shorter gradient times were used for some experiments and 0.1 % Formic acid was substituted for TFA for examples of MS operating conditions. A flow rate of 1 mL/min was used and peptide elution was monitored at 214 nm. Results and Discussion Key to understanding the performance improvements realized with higher efficiency Kinetex media is to compare peptide maps of a large protein where several peptides from the digested mixture are likely to not be fully resolved using existing 3 µm or 5 µm fully po- rous media. A direct comparison of a BSA tryptic digest run on the
Transcript of TN-1076 APPLICATIONS TN-1076 ... Mobile Phase: A: 0.1 % Trifluoroacetic acid in Water B: ... netex...
APPLICATIONSTN-1076
Page 1 of 4For additional technical notes, visit www.phenomenex.com
High Resolution LC/MS Analysis in Peptide Mapping Using Kinetex™ HPLC Columns Michael McGinley and Jeff LaynePhenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501 USA
The improved efficiency and resolution that Kinetex columns pro-vide is often used for decreasing chromatographic run times; for peptide mapping the improved performance of Kinetex columns can be used to get better resolution of closely eluting peptides. This improved peptide resolution allows one to better identify and quantitate low-level and post-translationally modified peptides. In addition to increased efficiency, Kinetex columns demonstrate good selectivity, porosity, and inertness, all of which have a critical impact on the usefulness of Kinetex columns for peptide mapping applications.
IntroductionReduced run time, leading to better throughput, is typically cited as the principle reason for using next generation HPLC columns (UHPLC, monolith, or core-shell). For small molecule separations, bioanalytical separations resolution of closely related peptides as well as improved sensitivity of low abundant components, are the key parameters of interest to most analytical biochemists.
Peptide mapping is the principle bioanalytical application where resolution is important; proteins are complex biopolymers that of-ten have post-translational modifications (PTMs) that can affect the activity and immunogenity of a protein therapeutic. Since modifica-tions are only a slight chemical change in the overall protein, pro-teins must be enzymatically fragmented into dozens or hundreds of peptides that are then separated by reversed phase HPLC and positively identified by LC/MS. Good resolution of closely related peptides is critical for identifying PTMs and next generation HPLC columns with higher efficiency have allowed for more accurate information from complex protein therapeutics like Ig-G proteins, which represent a majority of candidates currently in clinical trials.
However, high efficiency is not the only important parameter in peptide mapping of complex proteins. HPLC media must have high selectivity of minute chemical differences, have acceptable loading of analytical amounts of digested protein so that low level PTMs can be detected, and media must maintain good performance in MS-compatible buffers used for such analyses. Kinetex, a recently introduced core-shell media, was evaluated against popular fully porous media as well as other superficially porous media in re-gard to peptide mapping performance, peptide loading, and per-formance in volatile buffers and with Ig-G peptide separations to demonstrate the advantages of such a media over existing chro-matographic solutions.
ExperimentalRecombinant and natural proteins and peptides, dithiothreitol (DTT) and iodoacetic acid (IAA) were purchased from Sigma Chemicals (St. Louis, MO, USA). Recombinant human protein Interferon-alpha was obtained from Abcam Inc. (Cambridge, MA, USA)). Solvents were purchased from Fisher Scientific (Fairlawn, NJ, USA). Mobile phase modifiers and Asp-N endoproteinase were obtained from EMD (San Diego, CA). Lys-C endoproteinase was obtained from Wako Chemicals (San Diego, CA). Sequencing grade trypsin was purchased from Roche Applied Science (Indianapolis, IA, USA). Hu-man Ig-G2 was obtained from Dako (Carpentaria, CA). Kinetex 2.6 µm C18 columns (50 or 150 x 4.6 mm) were used for HPLC analysis (Phenomenex, Torrance, CA). HALO® C18 columns were obtained from Mac Mod (Chadds Ford, PA) and XBridge™ C18 columns were purchased from Waters® (Milford, MA).
For BSA digests, protein was digested with sequencing grade trypsin 1:50 E/S in 0.1 M NH4HCO3 pH 8.0 at 37 ˚C for 18 hours then quenched with TFA or Formic acid before injection. Differ-ent gradients of TFA/Water or Formic acid/Water from 5 to 45 % B (Acetonitrile) over 30 minutes were used based on the appli-cation shown. For peptide mixtures, fast gradients from 5 to 50 % in 5 minutes were used. HPLC analysis were performed on an Agilent 1100 HPLC equipped with an autosampler, column oven, and MWD using ChemStation software (Agilent, Santa Clara, CA). Shorter gradient times were used for some experiments and 0.1 % Formic acid was substituted for TFA for examples of MS operating conditions. A flow rate of 1 mL/min was used and peptide elution was monitored at 214 nm.
Results and DiscussionKey to understanding the performance improvements realized with higher efficiency Kinetex media is to compare peptide maps of a large protein where several peptides from the digested mixture are likely to not be fully resolved using existing 3 µm or 5 µm fully po-rous media. A direct comparison of a BSA tryptic digest run on the
APPLICATIONSTN-1076
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Kinetex 2.6 µm C18 column versus a fully porous 3.5 µm C18 col-umn using identical running conditions on a standard HPLC system is shown in Figure 1. The improved efficiency of the Kinetex core-shell column is readily apparent with significantly narrower peak widths and increased peak height versus the fully porous media. Upon closer inspection one can also see the better selectivity that the Kinetex media delivers over the gradient with over double the peak count (63 vs. 30) that the fully porous media delivers. Such results demonstrate the utility of the Kinetex column for peptide mapping.
While improved diffusion of the Kinetex core-shell particle delivers higher efficiency compared to a fully porous particle of a similar size, another relevant performance advantage that improved porosity of Kinetex offers relates to the performance of large peptides/ small proteins on core-shell media as shown in Figure 2. Small proteins above 10 kilodaltons (kDa), tend to result in badly tailing peaks on fully porous media, due to pore-related restrictions of large pep-
tides diffusing through the particle. Core-shell media like Kinetex have a shorter diffusion path which should reduce pore-related diffusion limitations. When proteins as large as 16 kilodaltons are analyzed on Kinetex columns, good efficiency and selectivity are still realized. This increased porosity is especially useful for peptide map applications for large proteins where some glycosylated pep-tides can be especially large.
Figure 2: Peptides on Kinetex 2.6 µm C18 Peptide run on a Kinetex 2.6 µm C18 column. Peptide larger than 10 kDa in size will typically start to broaden and tail on 100 Å columns and thus can give poor peak shape for peptide map applications of larger proteins. Note that both Cytochrome c (12 kDa) and Myoglobin (17 kDa) maintain good peak shape on the core-shell Kinetex column.
Figure 1. BSA Peptide Map Comparison HPLC Chromatogram of a tryptic digest of BSA comparing Kinetex to a fully porous 3.5 µm media. Note the increased peak capacity and peak height for the Kinetex column versus the fully porous media.
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Kinetex™ 2.6 µm C18
Conditions the same for both columns:Column: Kinetex 2.6 µm C18
XBridge 3.5 µm C18Dimensions: 150 x 4.6 mm
Mobile Phase: A: 0.1 % Trifluoroacetic acid in Water B: 0.1 % Trifluoroacetic acid in Acetonitrile
Time (min) % B0 330 50
Flow Rate: 1.2 mL/minTemperature: 40 °C
Detection: UV @ 214 nmSample: BSA tryptic map
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MET-Enkephalin (574)
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Angiotensin I (1,100)
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Insulin (5,700)
APPLICATIONSTN-1076
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Another relevant concern for any high-performance separation re-lates to the inertness of a media and its effect on the peak shape of peptides. Inertness of a media can be a significant concern when LC/MS compatible volatile (non-ion pairing) buffers are used. Trif-luoroacetic acid is an ion-pairing agent typically used as a mobile phase modifier for HPLC because it minimizes non-specific inter-actions between basic amino acids and acidic silanol groups that causes peak tailing and loss of resolution. While the ion-pairing affects of TFA improve peak shape, for LC/MS applications that same ion-pairing reduces electrospray efficiency resulting in poor sensitivity. Thus, for LC/MS peptide mapping applications, formic acid is often used as a mobile phase modifier instead to improve sensitivity. With less inert HPLC media this results in a significant peak tailing and loss of resolution; Figures 3A and 3B compare the inertness of Kinetex media over a different superficiously porous media for a peptide map of BSA using formic acid as the mobile phase modifier. In Figure 3A the peptide map on the Kinetex col-umn maintains good peak shape and peak capacity; for the com-petitor’s Fused-Core® column (Figure 3B) peak shape quality and peak capacity are dramatically less than the Kinetex column. Such
results demonstrate the utility of the Kinetex C18 column for pep-tide mapping applications, as LC/MS is often used to confirm the identity of each peptide in a map.
ConclusionsWith several “ultra-high performance” HPLC columns entering the market over the last few years, there have been many research-ers that have looked to such separation products as a means of improving the resolution and sensitivity of their peptide mapping applications. Increased efficiency can improve resolution of pep-tides in a protein digest, but such HPLC products also need to have good peptide selectivity and column inertness to be of utility for peptide mapping applications. Not only does a Kinetex 2.6 µm C18 column provide very high efficiency and peak capacities at standard HPLC backpressures, but Kinetex core-shell media also provides good separation of peptides, even using MS-compatible mobile phase buffers, when other less inert media underperform. The high porosity of Kinetex columns is of additional benefit for peptide mapping applications where larger peptides may be pres-ent in a digest mixture. Such characteristics make Kinetex columns an excellent solution for peptide mapping applications.
Conditions the same for all columns (except where noted):Column: Kinetex 2.6 µm C18
Dimensions: 50 x 4.6 mm Mobile Phase: A: 0.1 % Trifluoroacetic acid in Water
B: 0.1 % Trifluoroacetic acid in Acetonitrile
Time (min) % B0 55 95
Flow Rate: 2 mL/minTemperature: ambient
Detection: UV @ 214 nmSample: As noted
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Figures 3A and 3B: Kinetex vs. HALo HSA Peptide Map with Formic Acid Comparison of BSA peptide maps using MS-compatible conditions (0.1 % formic acid as modifier). The chromatogram in Figure 3A uses a Ki-netex 2.6 µm C18 column and 3B uses a HALO 2.7 µm C18 column. Note the significantly higher peak capacity for the Kinetex column and overall good peak shape versus the HALO column where several peak co-migrate and significant tailing. The inertness of the Kinetex column makes it com-patible with LC/MS conditions.
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Cytochrome c (12,000)
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Myoglobin (16,700)
Conditions the same for both columns:Column: Kinetex 2.6 µm C18
HALO 2.7 µm C18Dimensions: 150 x 4.6 mm
Mobile Phase: A: 0.1 % Formic acid in Water B: 0.1 % Formic acid in Acetonitrile
Time (min) % B0 330 50
Flow Rate: 1 mL/minTemperature: 35 °C
Detection: UV @ 214 nmSample: BSA tryptic digest
Gradient:
3B. HALo® 2.7 µm C18
3A. Kinetex™ 2.6 µm C18
APPLICATIONSTN-1076
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Terms and Conditions Subject to Phenomenex Standard Terms & Conditions, which may be viewed at www.phenomenex.com/TermsAndConditions
Trademarks Kinetex and KrudKatcher are trademarks of Phenomenex, Inc. HALO and Fused-Core are registered trademarks of Advanced Materials Technology, Inc. XBridge is a trademark of Waters Corporation.
Disclaimer Phenomenex is in no way affiliated with Advanced Materials Technology or Waters Corpora-tion. Comparative separations may not be representative of all applications.
© 2010 Phenomenex, Inc. All rights reserved.
If Phenomenex products in this technical note do not provide at least an equivalent separation as compared to other products of the same phase and dimensions, return the product with compara-tive data within 45 days for a FULL REFUND.
KrudKatcher Ultra In-Line Filter ordering InformationPart No. Description UnitAF0-8497 KrudKatcher Ultra In-Line Filter,
0.5 µm Porosity x 0.004 in. ID3/pk
KrudKatcher Ultra requires 5/16 in. wrench. Installation wrench not provided.
KrudKatcher™ Ultra In-line Filter The KrudKatcher Ultra filter body houses an integrated 0.5 µm 316 stainless steel filter element that efficiently removes microparticu-lates from the flow stream without contributing to system back-pressure or dead volume (<0.2 µL).
2.6 μm Minibore Columns (mm)Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1C18 00A-4462-AN 00B-4462-AN 00D-4462-AN 00F-4462-ANPFP –– 00B-4477-AN 00D-4477-AN 00F-4477-ANHILIC –– 00B-4461-AN 00D-4461-AN 00F-4461-AN
2.6 μm Solvent Saver MidBore™ Columns (mm)Phases 50 x 3.0 100 x 3.0 150 x 3.0C18 00B-4462-Y0 00D-4462-Y0 00F-4462-Y0PFP 00B-4477-Y0 00D-4477-Y0 00F-4477-Y0HILIC –– –– 00F-4461-Y0
2.6 μm Analytical Columns (mm)Phases 30 x 4.6 50 x 4.6 100 x 4.6 150 x 4.6C18 00A-4462-E0 00B-4462-E0 00D-4462-E0 00F-4462-E0PFP –– 00B-4477-E0 00D-4477-E0 00F-4477-E0HILIC –– 00B-4461-E0 00D-4461-E0 00F-4461-E0
Kinetex ordering Information1.7 μm Minibore Columns (mm)Phases 50 x 2.1 100 x 2.1 150 x 2.1C18 00B-4475-AN 00D-4475-AN 00F-4475-ANPFP 00B-4476-AN 00D-4476-AN 00F-4476-ANHILIC 00B-4474-AN –– ––
