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ADVANCED TOPICS IN PEPTIDE MAPPING: DEVELOPING REPRODUCIBLE HIGH RESOLUTION SEPARATIONS

Authors: P. Jayaraman1, S. Koza1, C. Reed1, H. Yang1, E. Chambers1 1Waters Corporation, Consumables Group, Milford, USA.

INTRODUCTION The peptide mapping of proteins is a fundamental tool used that

has been applied to both the proteomics-based discovery of new

biotherapeutic proteins and for monitoring the modification and

degradation of those proteins as they are developed and

commercialized1. When developing a reproducible and informative

peptide map the enzymatic digestion protocol and the separation

of the resultant peptides need to be optimized. We will focus on

column selection considerations for reversed-phase separation

based peptide mapping, however, many of these column selection

considerations can also be directly applied to the selection of

columns for LC-MS based proteomics studies and synthetic

peptide purity analyses.

Ten distinctly different Waters RP column types, representing

variations in ligand characteristics, base particle composition, and

particle size, were evaluated with acetonitrile gradients with either

0.1% trifluoroacetic acid (TFA) or 0.1% formic acid (FA) as an ion-

pairing reagent. Separations of both a peptide standard mix and

the tryptic digest of a reference monoclonal antibody were used for

this comaparison. The performance metrics for this evaluation

included both peak capacity (PC) and peptide retention. In addition

several of the selectivity differences observed between selected

columns are also highlighted.

METHODS SAMPLE DESCRIPTION: MassPrep Peptide Mixture (Product Number: 186002337 sample was reconstituted in 0.1% formic acid to a concentration of ~15 µg/mL per peptide. NIST monoclonal antibody (NISTmAb) reference material, RM 8671 was reduced and alkylated (iodoacetamide). The sample was acidified prior to analysis with 1:9 ratio of 1% formic acid. The final concentration of injected mAb sample was ~ 0.1 mg/mL.

MassPREP Peptide Standard

mAB Peptides

METHOD CONDITIONS (unless noted otherwise):

LC System: Waters ACQUITY UPLC H-Class Bio System Detection: ACQUITY UPLC TUV detector, 5mm Titanium flow cell Wavelength: 214nm , Column Temp.: 60 °C, Sample Temp.: 10 °C Injection Volume: 15 µL for MassPrep Peptide Mixture and 50 µL for NIST mAB digest Flow Rate: 0.2 mL/min, A: 0.1% (v/v) trifluoroacetic acid (TFA) or formic acid (FA) in water B: 0.1% (v/v) TFA or FA in acetonitrile Gradient: from 0.5 to 50%B in 30 or 60 minutes

Columns (all 2.1mm X 150 mm)

ACQUITY UPLC Peptide BEH C18 Column, 130Å, 1.7 µm

ACQUITY UPLC Peptide CSH C18 Column, 130Å, 1.7 µm

XSelect Peptide CSH C18 XP Column, 130Å, 2.5 µm

ACQUITY UPLC Peptide BEH C18 Column, 300Å, 1.7 µm

ACQUITY UPLC Peptide HSS T3 Column, 100Å, 1.8 µm

ACQUITY UPLC CSH Phenyl-Hexyl Column, 130Å, 1.7 µm

CORTECS C18 Column, 90Å, 2.7 µm

CORTECS UPLC C18+ Column, 90Å, 1.6 µm

CORTECS C18+ Column, 90Å, 2.7 µm

CORTECS UPLC T3 Column, 120Å, 1.6 µm

Chromatography Software: Waters Masslynx (v 4.1) and Waters UNIFI (v 1.8 )

Mass spectrometer: Xevo G2 Q-Tof:

Ionization mode: ESI+, Analyzer mode: Resolution, Scan rate: 2 Hz

Capillary voltage: 3.00 kV, Cone voltage: 62 V, Source temp.: 150 °C

Desolvation temp.: 450 °C, Cone gas flow: 2.0 L/h,

Desolvation gas flow: 600 L/h, Calibration: NaI from 100 to 2000 m/z

Acquisition: 100 to 2000 m/z, 10 Hz scan rate

RESULTS (CONTINUED) Peptide Retention Summary

The USP General Chapter <1055> states that 95% or greater sequence coverage be the target for a peptide map. Thus, a RP column that provides greater peptide retention can be useful if the map contains numerous hydrophilic peptides. For this evaluation the calculated amounts of acetonitrile at which the four least hydrophobic peptides in the MassPREP Peptide mixture eluted were used to evaluate retentiion. All peptides were well retained in 0.1% TFA (data not shown), including RASG-1, which is predicted to have a retention factor (k') of 10 at acetonitrile concentrations of only 4.40% and 0.54% in TFA and FA with a 100Å C18 column (SSRCalc, v Q)

1. The data obtained for 0.1%

FA are shown in Figure 5. The trend in retention among the columns with 0.1% TFA was comparable to that observed for 0.1% FA. The Peptide HSS T3, Peptide BEH C18 130Å and 300Å, and CORTECS T3 columns provided the highest retentivities.

Peptide Selectivity Summary

Selectivity differences among phases can also be used to an advantage to improve the resolution of a critical pair of closely eluting peptides during the development of a peptide mapping Selectivity differences can be the result of surface chemistry differences (Figure 6) and changes in characteristics of the base particle such as pore size (data not shown).

CONCLUSION All of the Waters RP columns evaluated are potentially capable of producing an effective peptide mapping separation. However, screening all of these columns along with columns from other manufacturers is not feasible. Therefore, a subset of four of these Waters RP particle technologies that are the most recommended for the successful development of RP peptide mapping separations have been identified and their attributes summarized. In addition,

RESULTS Selected examples of the separations achieved for the peptide standards used in this study are shown in Figures 1. Additionally overlays of the peptide maps in both TFA and FA are shown in Figure 2.

Peak Capacity Summary

PC is a measure of the separation capability of a column and is representative of the average resolution that a column can provide in a a complex separation such as a peptide map. PC values are dependent on sample load , therefore, in these experiments peptide loads typically used for peptide mapping analysis of a biotherapeutic protein were employed. These higher loads provide the dynamic range required to reliably monitor low abundance modified peptides. Comparisons of the PC results for all of the columns evaluated with the MASSPrep Peptide standard are presented in Figure 3 (0.1% TFA) and Figure 4 (0.1% FA), The PC values determined for the individual peptides do not always follow the average trends. These variations are the result of specific peptide characteristics (e.g. charge, hydrophobicity, size) and the surface chemistry and morphology (pore size) of the RP particle. For example, it was observed that as peptide sizes approached ~2000 MW the 300Å pore size BEH C18 column PC is slightly higher than that of the 130Å BEH C18 column (data not shown).

Peak Peptide Molecular Weight (mono-isotopic)

pI Sequence

1 RASG-1 1000.49 9.34 RGDSPASSKP

2 angiotensin frag. (1-7) 898.47 7.35 DRVYIHP

3 bradykinin 1059.56 12.00 RPPGFSPFR

4 angiotensin II 1045.53 7.35 DRVYIHPF

5 angiotensin I 1295.68 7.51 DRVYIHPFHL

6 renin substrate 1757.93 7.61 DRVYIHPFHLLVYS

7 enolase T35 1871.96 7.34 WLTGPQLADLYHSLMK

8 enolase T37 2827.28 3.97 YPIVSIEDPFAEDDWEAWSHFFK

9 melittin 2845.74 12.06 GIGAVLKVLTTGLPALIS-WIKRKRQQ

Peptide Molecular Weight (monoisotopic)

pI Sequence

H31 447.27 9.35 TISK

10 699.43 9.35 NQVVLK

H14 1321.65 8.63

STSGGTAALGCLVK

L15 1501.75 6.18 DSTYSLSSTLTLSK

H23 1676.79 5.53 FNWYVDGVEVHNAK

H38 1872.91 4.10 TTPPVLDSDGSFFLYSK

H37 2543.12 3.96 GFYPSDIAVEWESNGQPENNYK

L7 4482.98 3.94

FSGSGSGTEFTLTISSLQPDDFATY-YCFQGSGYPFTFGGGTK

H15 6713.29 7.46 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK

Figure 1. Comparison of highest and lowest PC separations for MASSPrep Peptide standards using 0.1% TFA or 0.1% FA in water/

Figure 2. Comparison of mAb peptide map separations with 0.1% TFA .Peak identified with asterisk (*) is produced by the reduction/

Figure 3. Comparison of PC results for MASSPrep Peptide standards using 0.1% TFA .

Figure 4. Comparison of PC results for MASSPrep Peptide standards using 0.1% FA .Missing PC values (*) for melittin and emolase TT35 peptides for CSH Phenyl-Hexyl column due to co-elution of peptides.

Figure 5. Comparison of calculated peptide retention results determined for MASSPrep Peptide standards using 0.1% FA in water/acetonitrile.

Figure 6. XIC profiles showing the effect of particle surface charge on selectivities for NIST mAB tryptic peptides (0.1% TFA) . Chromatograms have been aligned to the retention time of the H38.

Particle Technology Recommended UPLC and HPLC

Particle Sizes (µm)

Comments

Mo

st

Reco

mm

en

ded

Peptide CSH C18, 130Å 1.7, 2.5 Highest PC in both TFA and FA mobile phases and

significant differences in selectivity compared to

BEH C18 phases. Peptide retention is lower than

HSS T3 and BEH C18 phases.

Peptide BEH C18, 130Å 1.7, 3.5 High PC in both TFA and FA and high peptide

retention.

Peptide BEH C18, 300Å 1.7, 3.5 High PC in TFA ,moderate PC in FA, and moderate

peptide retention. Slightly improved PC for larger

MW peptides.

Peptide HSS T3, 100Å 1.8, 2.5 Highest peptide retention and moderate to high

PC in both TFA and FA. Slightly diminished PC for

larger MW peptides.

Alt

ern

ati

ves

CORTECS T3 120Å 1.6, 2.7 High PC in TFA ,moderate to low PC in FA, and

high peptide retention.

CSH Phenyl-Hexyl, 130Å 1.7, 2.5 High PC in both TFA and FA mobile phases and

significant differences in selectivity. Peptide

retention is significantly lower than other phases

tested.

CORTECS C18+, 90Å 1.6, 2.7 High PC in TFA and FA, low peptide retention.

Smaller pore size not as amenable to larger

peptides.

CORTECS C18, 90Å 1.6, 2.7 High PC in TFA but had the lowest PC in FA,

moderate peptide retention. Smaller pore size

not as amenable to larger peptides.

References

1. Krokhin OV, Spicer V. Peptide retention standards and hydrophobicity indexes in reversed-phase high-performance liquid chromatography of peptides. Analytical chemistry. 2009; 81:9522-30.