This application note demonstrates the rapid separation of a wide mass range of peptides and ... transfer observed at elevated flow rates in porous media. This unique ... Thermo Scientific⢠Dionex⢠UltiMate⢠3000 RSLCnano system.
Shane Bechler, Kelly Flook, Thermo Fisher Scientific, Sunnyvale, CA, USA
Appli cat i on N ote 2 0 9 7 2
Rapid Separation and Mass Spectrometry Detection of Intact Protein Samples Using Monolithic Capillary Columns
Key Words Proteins, ProSwift, RP-4H, reversed phase, proteomics, mass spectrometry, high throughput, biomolecules, capillary column, monolith, biomolecules, top-down proteomics
Abstract This application note demonstrates the rapid separation of a wide mass range of peptides and proteins using a reversed-phase monolithic column with both UV and MS detection. The separation is conducted on Thermo Scientific™ ProSwift™ RP-4H columns using conventional water/ acetonitrile-based eluents. These simple, straightforward methods can be used to optimize chromatographic separations for analysis time, resolution, and MS detection especially for top-down proteomics.
Introduction ProSwift RP-4H monolith columns use an in situ polymerization process to create a continuous polymer rod possessing a network of interconnected pores. The poly(divinylbenzene-co-ethylvinylbenzene) chemistry of the polymeric monolith makes it useful for reversed-phase separation of a wide range of biomolecules. When running a gradient from low to high organic concentration, hydrophobic biomolecules that bind to the column media under aqueous conditions will desorb and elute with increasing organic content. This allows the user to control the chromatographic separation by adjusting the gradient. Optimizing the gradient is particularly useful when analyzing complex mixtures containing hundreds or thousands of biomolecules. With monolithic polymeric columns, there is minimal diffusion related band broadening; therefore, after the protein molecule has desorbed from the surface, increased flow rates result in not only faster analysis but also narrow peak widths and increased peak capacity. The separation and analysis of proteins is of great importance in the fields of proteomics, biomarker discovery, clinical research, and systems biology. Conventional methods for this work include the separation of a complex mixture of proteins on a reversed-phase liquid chromatography column coupled to a mass spectrometry instrument (LC-MS), for the characterization and identification of individual proteins.
HPLC columns packed with non-porous beads display excellent mass transfer, which results in excellent resolution of analytes. However, the smaller particle packed bed columns commonly operate at high pressure and can be easily fouled when analyzing complex samples of large proteins. Additionally, non-porous beads suffer from loading limitations, consequently compromising protein separation and reducing column lifetime. In contrast, monolithic columns possess both relatively large through channels, which reduce operational backpressure, and smaller through pores for efficient analyte separation. As the proteins pass through these continuous small pores they are not hindered or subject to poor diffusive mass transfer observed at elevated flow rates in porous media. This unique combination of pore sizes enables these columns to efficiently separate proteins at high flow rates for fast analysis, while maintaining a low column
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backpressure to allow for room temperature operation. Additionally, the low sample carryover results in reduced fouling of the stationary phase and an extended column lifetime. For these reasons ProSwift RP-4H monolithic columns are an excellent choice for the rapid analysis of complex and high molecular weight protein samples. This application note illustrates how changing the flow rate, and essentially adjusting the gradient, can improve the separation of a simple mixture and be used to increase sample throughput and/or improve peak capacity. Additionally, it demonstrates the use of these capillary columns in combination with MS to separate, identify, and characterize hundreds of proteins in one sample.
Experimental Details Consumables
Part Number
Fisher Scientific™ LC/MS grade water
W/011217
Fisher Scientific LC/MS grade acetonitrile
A/0638/17
Fisher Scientific Analytical grade formic acid
F/1900/PB08
Thermo Scientific™ Pierce™ Trifluoroacetic acid LC/MS grade
28901
Thermo Scientific MS Certified Convenience Kit, Clear 9 mm Wide Opening Screw Vial, 2 mL Screw Thread Closure with Bonded Clear PTFE/Clear Silicone Septum
MSCERT4000-34W
ProSwift RP-4H Monolithic Nano Column, 200 µm ID × 25 cm
164923
ProSwift RP-4H Monolithic Nano Column, 100 µm ID × 50 cm
164921
Sample Preparation Sample for Figure 1 Compounds:
Angiotensin II, substance P, lysozyme, myoglobin, l-lactic dehydrogenase, each 5 µg/mL
Water + 0.1% trifluoroacetic acid (TFA)
Matrix:
Sample for Figure 2
HeLa cell lysate molecular weight fractions: 11–23 kDa and 20–52 kDa
Separation Conditions
Instrumentation:
Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano system
Flow Selector:
For Capillary LC with Flow Range 0.5-10 µL/min
Column:
(1) ProSwift RP-4H Monolithic Nano Column, 200 µm ID × 25 cm (2) ProSwift RP-4H Monolithic Nano Column, 100 µm ID × 50 cm
Gradient conditions for Figure 1
Mobile phase A:
Water / acetonitrile (95:5 v/v) + 0.1% trifluoroacetic acid
Mobile phase B:
Water / acetonitrile (5:95 v/v) + 0.1% trifluoroacetic acid
Gradient:
Refer to Table 1
Flow rate:
2, 4, 8 µL/min
Column temperature:
35 °C
Injection details:
1 µL
Injection wash solvent 1:
Water / isopropyl alcohol (98:2 v/v)
Time (min)
%A
%B
-5.0
99
1
0
99
1
0.1
99
1
10.1
35
65
10.2
1
99
13.2
1
99
13.3
99
1
16.3
99
1
Table 1: LC gradient conditions for Figure 1
6041.0003
3
Gradient Conditions for MS
Mobile phase A:
Water / acetonitrile (95:5 v/v) + 0.2% formic acid
Mobile phase B:
Water / acetonitrile (5:95 v/v) + 0.2% formic acid
Gradient:
Refer to Table 2
Flow rate:
1 µL/min
Time (min)
%A
%B
0
95
5
Valve 1_10 (loading)
5
95
5
MS acquisition start Valve 1_2 (inject)
15
95
5
16
95
5
66
45
55
67
2
98
70
2
98
71
95
5
84
95
5
Comment
Table 2: LC gradient conditions for Figure 2
MS Conditions The Thermo Scientific™ Orbitrap Elite™ hybrid ion trap-Orbitrap mass spectrometer was used for analysis. The MS parameter settings are provided in Table 3.
Parameter
Setting for 11−23 kDa Fraction
Setting for 20−52 kDa Fraction
Ion source
Nano-ESI
Nano-ESI
Instrument control software
Tune 2.7
Tune 2.7
Capillary temperature (°C)
320
320
S-lens RF voltage
50%
50%
Source voltage (kV)
1.8
1.8
500–2000
500–2000
1 × 106
3 × 104
2000
50
Microscans
4
25
SID (V)
15
15
Full MS mass range (m/z) Full MS parameters Target value Max injection time (ms)
MS2 parameters Resolution settings (FWHM at m/z 400)
60,000
60,000
Target value
1 × 106
1 × 106
Isolation width (Da)
50
50
Minimum signal required
500
500
Normalized collision energy (HCD)
25
25
Activation time (ms)
0.1
0.1
2
2
Lowest m/z acquired
400
400
Max injection time (ms)
2000
2000
Top-N MS2
Table 3: Orbitrap Elite MS settings for data collection of 11–23 kDa and 20–52 kDa MW fractions from HeLa cell lysate
Data Processing Software:
Thermo Scientific™ Dionex™ Chromeleon™ software version 6.8
Thermo Scientific™ Xcalibur™ software (No DCMS-Link)
Thermo Scientific™ ProSightPC software was used to search the data against a Homo sapiens taxonomy subset of the Swiss Prot database.
Results To assess a wide range of molecular weights, the analysis of a simple peptide and protein mixture was performed on a 200 µm × 25 cm ProSwift RP-4H column at three different flow rates while maintaining a constant gradient time. Figure 1 shows the chromatographic separation of this mixture. As the flow rate increased the selectivity of the peaks did not change. However, at elevated flow rates the analytes eluted from the column with less influence of diffusion; the decrease in peak widths resulted in improved resolution. Additionally, increased flow rates afforded decreased gradient delay time resulting in an overall shorter analysis time. When analyzing a large number of samples, these small changes in separation time can significantly reduce the amount of time required to complete analysis. The ability to achieve narrower peaks can significantly increase the peak capacity of the method. At 2, 4, and 8 µL/min, the peak capacities calculated using the average peak widths of the 5 species over the 10 minute gradient window were determined to be 47, 72, and 95, respectively, approximately doubling from 2 to 8 µL/min. This is a significant increase in peak capacity simply by increasing the flow rate without adjusting the length of the gradient. It should also be noted that as the gradient time was not adjusted, the analytes eluted from the column at the same time in the gradient. However, by increasing the flow rate, the gradient delay was reduced as is the time it takes the eluted protein to exit the column. The overall effect is approximately a 20% decrease in analysis time by increasing the flow rate from 2 to 8 µL/min. The effect of increased flow rate is especially relevant during tandem applications using valve switching.
12.0
3
1 2
2 µL/min
4 5
-2.0 10.0
3
4 µL/min
1 mAU
4
4
2
5
-2.0 8.0
Column: Eluents: Gradient: Flow Rate: Inj. Volume: Temp.: Detection: Sample:
ProSwift RP-4H 200 µm x 25 cm A: water/acetonitrile (95/5 v/v) + 0.1% trifluoroacetic acid B: water/acetonitrile (5/95 v/v) + 0.1% trifluoroacetic acid 1–65% B in 10 minutes (5 minute pre-run equilibration) 2, 4, and 8 µL/min 1 µL 35 °C UV at 214 nm Peptides and proteins each at 5 µg/mL in DI water + 0.1% trifluoroacetic acid
Peaks:
1. Angiotensin II 2. Substance P 3. Lysozyme 4. Myoglobin 5. L-Lactic Dehydrogenase
8 µL/min
3 1 4
2
5 -1.0 0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
19.0
Time, min
Figure 1: The effect of flow rate using the same gradients on the ProSwift RP-4H, 200 µm x 25 cm monolith column
Calculations Peak Capacity ≈
tG W
+1
where tG is the gradient time and W is the average peak width at 13.4% height
48kDa
6,061 Da 11,470 Da
14,902 Da
11-23 kDa 0
10
20
30
40
50
60
Time (min)
70
80
90
100
110
20-52 kDa
120
0
56kDa
Time (min)
120
42.6kDa 11.4kDa
12.0kDa
46.8kDa 35.8kDa
17.2kDa
45.9kDa
17.8kDa 18.9kDa
47.7kDa
20.8kDa 17.3kDa
14.4kDa
51.8kDa 20.7kDa 21.8kDa 18.4kDa
16.0kDa 20.9kDa 45.8kDa
31.5kDa 16.8kDa
20.7kDa
22.8kDa
10
Time (min)
70
Time (min)
10
Figure 2: Base peak chromatograms showing MS detection of HeLa cell lysate fractions on the ProSwift RP-4H, 100 µm x 50 cm column. Data courtesy of Prof. Neil Kelleher, Northwestern University.
Conclusion •
Using a constant gradient time and increasing the flow rate results in increased peak capacity and resolution, along with decreased peak widths and gradient delay time. Increasing the flow rate can therefore be used to optimize the separation of proteins and reduce the time required for analysis of large sample sets.
• ProSwift RP-4H capillary columns are well suited for the separation of protein samples for the purposes of MS detection and characterization.
For research use only
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Appli cat i on N ote 2 0 9 7 2
The capillary ProSwift RP-4H columns are specifically designed to be used at flow rates suitable for 14,038 Da MS detection of proteins. Figure 2 demonstrates MS detection of a complex protein mixture separated on a ProSwift RP-4H 100 µm × 50 cm column. With gradient optimization and MS detection, the ProSwift RP-4H columns can be used to detect and characterize hundreds of species when analyzing complex samples.
