AbstractMethods for LC-MS peptide analysis often use acidified mobile phases, which can alter retention and selectivity. Here, we demonstrate the effects of modifying acid type, acid concentration, temperature, and gradient slope using several test probes. A Raptor ARC-18 col-umn was employed for this work because the stationary phase ligand is sterically protected, making it extremely stable and resistant to acid damage at low pHs, which is an important characteristic when developing LC-MS methods for peptide analysis.

IntroductionWith the influx of biotherapeutics in medical research and healthcare, LC-MS analysis of peptides and small proteins continues to grow. Many of these analyses utilize acid-modified mobile phases to improve peak shape [1]; however, the effects of acidic mobile phases on selectivity and retention are often not well understood. In this article, we will explore the effects of acid type, acid concentration, temperature, and gradient slope on the selectivity and retention of several peptide probes and one small protein probe (cytochrome C) using the Raptor ARC-18 LC column. This column was selected because the ligand is sterically protected from acid damage and is stable to pH 1.0. In addition, it contains superficially porous particles (SPP), which allow for faster analysis times than can be achieved with fully porous particle (FPP) columns.

ExperimentalSeveral experiments were carried out to demonstrate the effects of acid-modified mobile phases, temperature, and gradient slope on LC-MS peptide analysis. For this work, the peptide and small protein standards in Table I and a bovine serum albumin (BSA) trypsin digest standard (Protea) were diluted in acidified water. These standards were injected into a Waters ACQUITY UPLC H-Class system configured with a PDA and QDa for simultaneous UV and MS detection. A wavelength of 215 nm and electrospray ionization in positive ion mode was used for UV and MS detection, respectively. Reversed-phase separations were performed using water and acetonitrile mobile phases modified with 0.01, 0.05, and 0.1% formic acid (FA) or trifluoroacetic acid (TFA) under gradient conditions using a Restek Raptor ARC-18 LC column. Specific conditions for each experiment are presented in Figures 2-5.

Influencing Selectivity in LC-MS Peptides Analysis on the Raptor ARC-18 Column

By Sharon Lupo and Frances Carroll

Figure 1: Raptor ARC-18 LC Column Properties.

SiO

Stationary Phase Category:C18, octadecylsilane (L1)

Ligand Type: Sterically protected C18

Particle: 2.7 μm or 5 μm superficially porous silica (SPP or “core-shell”)

Pore Size: 90 Å

Surface Area: 150 m2/g (2.7 μm) or 100 m2/g (5 μm)

Recommended Usage:pH Range: 1.0–8.0

Maximum Temperature: 80 °C

Maximum Pressure: 600 bar / 8,700 psi (2.7 μm) or 400 bar / 5,800 psi (5 μm)

Properties:• Well-balanced retention profile.

• Sterically protected to resist harsh, low-pH mobile phases.

• Ideal for use with sensitive detectors like mass spec.

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Results and DiscussionSeveral probes were used to aid in the understanding of selectivity changes that affect LC-MS peptide analyses when the type and concentration of the acidic modifier in the mobile phase is manipulated. The retention of peptides containing basic amino acids (e.g., arginine) exhibited considerably large changes in retention when switching between formic acid and TFA. In Figure 2, the peptide MET-ARG-PHE-ALA not only changed elution order but also displayed improved peak shape and was more retained due to the increased ion pairing capabilities of TFA over formic acid. Alternatively, VAL-TYR-VAL is a polar peptide with an overall neutral charge whose changes in selectivity result from the favorable decrease in mobile phase pH when TFA is used; in this case, the peptide was less ionized and therefore better retained. Variations in acid concentration had a comparable effect. In Figure 3, overall retention times increased with increasing concentrations of TFA, and improvements in peak shape were observed for the small protein, cytochrome C (12.3 kDa). For this reason, sterically protected columns that are stable at low pH, such as the Raptor ARC-18 column, are often preferred for LC-MS peptide analysis.

Table I: Probes used for LC-MS Peptide Analyses

Peak ID Compound Molecular Weight (Da) SIR (m/z)

1 GLY-TYR 238 239.3

2 VAL-TYR-VAL 380 380.5

3 MET-ARG-PHE-ALA 524 524.6

4 Methionine enkephalin 574 574.6

5 Leucine enkephalin 556 556.7

6 Angiotensin II 1,046 524.4

7 Neurotensin 1,700 837.9

8 Cytochrome C 12,300 1,020.6

Figure 2: Effects of Acid Type on Selectivity and Peak Shape.

Time (min)0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

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0.1% FA

0.1% TFA

LC_BA0353

Column Raptor ARC-18 (cat.# 9314A12)Dimensions: 100 mm x 2.1 mm IDParticle Size: 2.7 µmPore Size: 90 ÅTemp.: 60 °CSample Custom peptide/small protein standard mixDiluent: 0.1% Formic acid in waterConc.: 0.2–0.4 mg/mLInj. Vol.: 2 µL

Mobile PhaseA: Water + acidB: Acetonitrile + acid Time (min) Flow (mL/min) %A %B 0.00 1 95 5 1.00 1 95 5 10.0 1 50 50 10.1 1 95 5 13.0 1 95 5

Detector MSIon Mode: ESI+Mode: SRMInstrument UHPLC

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Figure 3: Effects of Acid Concentration on Retention and Selectivity.

LC_GN0566

Column Raptor ARC-18 (cat.# 9314A12)Dimensions: 100 mm x 2.1 mm IDParticle Size: 2.7 µmPore Size: 90 ÅTemp.: 60 °CSample Custom peptide/small protein standard mixDiluent: Water + 0.1% formic acidConc.: 0.2–0.4 mg/mLInj. Vol.: 2 µLMobile Phase A: Water + TFAB: Acetonitrile + TFA Time (min) Flow (mL/min) %A %B 0.00 1 95 5 1.00 1 95 5 10.0 1 50 50 10.1 1 95 5 13.0 1 95 5 Detector MSIon Mode: ESI+Mode: MS scanInstrument UHPLC

0.500.00 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

0.500.00 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

0.500.00 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

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0.01% TFA

0.05% TFA

0.1% TFA

Time (min)

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Figure 4: Effects of Temperature on the Selectivity of Peptides in a BSA Tryptic Digest Standard.

In a second set of experiments, resolution and elution order changes were examined when temperature and gradient modifications were made to the analysis of a bovine serum albumin (BSA) trypsin digest standard. In the resulting peptide maps shown in Figure 4, base peak mass 468 m/z was resolved from an adjacent peak when a lower column oven temperature (40 °C) was used. Similarly, the gradient slope was shown to impact the band spacing of closely eluting compounds. This is demonstrated in Figure 5 where the number of resolved peaks increased with decreasing gradient slope. In general, steeper gradients can be used for the separation of specific peptides in order to achieve short cycle times. However, if numerous peptides from a digest are to be analyzed, a shallower gradient is typically preferred.

LC_GN0567

396

40 °C

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2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00Time (min)

60 °C

Column Raptor ARC-18 (cat.# 9314A12)Dimensions: 100 mm x 2.1 mm IDParticle Size: 2.7 µmPore Size: 90 ÅSample Bovine serum albumin (BSA) tryptic digest standardDiluent: Water + 0.1% formic acidConc.: 5 pmolInj. Vol.: 2 µLMobile Phase A: Water + 0.1% formic acidB: Acetonitrile + 0.1% formic acid Time (min) Flow (mL/min) %A %B 0.00 0.3 98 2 40.0 0.3 75 25 40.1 0.3 98 2 50.0 0.3 98 2Detector MSIon Mode: ESI+Mode: MS scanInstrument UHPLC

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Figure 5: Effects of Gradient Steepness on the Selectivity of Peptides in a BSA Tryptic Digest Standard.

535 53

5424

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2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

1.00 2.00 3.00 4.00 15.00 16.00 17.00 18.00 19.00 20.005.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

Time (min)

20 min GradientSlope = 1.15%

40 min GradientSlope = 0.575%

60 min GradientSlope = 0.383%

LC_GN0568

Column Raptor ARC-18 (cat.# 9314A12)Dimensions: 100 mm x 2.1 mm IDParticle Size: 2.7 µmPore Size: 90 ÅTemp.: 40 °CSample Bovine serum albumin (BSA) tryptic digest standardDiluent: Water + 0.1% formic acidConc.: 5 pmolInj. Vol.: 2 µL

Mobile Phase A: Water + 0.1% formic acidB: Acetonitrile + 0.1% formic acid Flow (mL/min) %A %B 0.3 98 2 0.3 75 25 0.3 98 2 0.3 98 2Detector MSIon Mode: ESI+Mode: MS scanInstrument UHPLC

ConclusionRelatively small changes in chromatographic parameters can have a large impact on LC-MS peptide analysis. In this work, we demonstrated that significant shifts in selectivity and retention can occur in response to relatively small changes to mobile phase acid (type and concentration), temperature, and gradient slope. Sterically protected columns like the Raptor ARC-18 column are stable at low pH and provide the versatility required for the development of peptide applications.

References[1] E. Wang, D. Kalu Appulage, F. Carroll, K. Schug, Intact protein separation and quantitation in biological fluids using HPLC and triple quadrupole mass spectrometry, ASMS, St. Louis, MO, 2015.

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