Reduction of Run Time in Peptide Mapping

This application note describes the reduction of run time in peptide mapping using BioSuite Columns.

Benefits

• A reduction in run time of peptide maps
• No reduction in peak capacity
• Maintenance of baseline resolution between critical pairs of peptides in a separation

A successful peptide map of a protein pharmaceutical is the result of manipulation and optimization of many chromatographic conditions. These factors include: flow rate, temperature, gradient slope, and duration, pH, mobile phase, and column. An important column characteristic that affects the separation of peptides is particle size. Decreased particle size enables separations with higher resolution and peak capacity. Currently, many peptide maps of protein pharmaceuticals are separated on columns containing 5 μm particles that are 250 mm in length. These columns have similar peak capacity as columns packed with 3 μm particles and 150 mm length. Therefore, using a column with similar peak capacity that is shorter in length enables reduced run time while maintaining resolution.

Protein Digestion Sample Preparation

The Waters MassPREP Enolase Digestion Standard (p/n: 186002325) was used in this analysis. One vial of the digestion standard was diluted in 100 μL of mobile phase A to obtain the appropriate working standard. The resulting protein digest contains no undigested enolase, trypsin or other hydrophilic components.

Run time, resolution of critical pairs, and peak capacity were examined in this study. Figure 1 shows the reduction in run time from 116 minutes using the 5 μm, 250 mm column to 70 minutes using the 3 μm, 150 mm column. This represents a 40 percent reduction in the run time of the peptide map. The data further shows the maintenance of good resolution when using the shorter, 150 mm, column. This is shown by the baseline resolution between a critical pair of peptides (Figure 1, inset). Finally, the peak capacity of each column was calculated. The peak capacity (Pc) of a column is defined as the number of peaks a column can separate within a given gradient time and was calculated by using the equation: 

P_c = 1+ (t_g / W_0.5h)

Where t_g represents the time of the gradient in minutes and W_0.5h represents the width of the peak at half height in minutes. Five peaks corresponding to five different peptides were selected from each column (Figure 2). Peak widths were then calculated, averaged and the peak capacity of each column was calculated (Table 1) The results of this calculation show equal peak capacities for both columns; 583 for the 3 μm, 150 mm column and 609 for the 5 μm, 250 mm.

The results of this study show that the use of shorter columns that contain smaller particles results in the following: 

1. A reduction in run time of peptide maps, a reduction of 40 percent was obtained in this study.
2. No reduction in peak capacity, the same peak capacity was obtained in this study using a shorter column packed with small particles (150 mm, 3 μm) as with the longer column packed with larger particles (250 mm, 5 μm).
3. Maintenance of baseline resolution between critical pairs of peptides in a separation. Therefore, one can significantly reduce the analysis time of peptide mapping without losing resolution of critical pairs and peak capacity by using the Waters BioSuite C₁₈, 3 μm, 150 mm, PA-A Column.