TSQ Quantiva's excellent sensitivity enhances the ability to quantify target peptides in complex matrices

Li Jing

Thermo Fisher Scientific (China) Co., Ltd.

Key words

TSQ Quantiva; peptide quantification; sensitivity

Foreword

Biomarker research includes Discovery, Verification, Validation, and finally into deeper stages such as Clinical Utilization. In the process of studying biomarkers, the main challenge is how to find medium-abundance or low-abundance proteins in complex biological samples, which puts high demands on the performance of the instrument, that is, the instrument must have high sensitivity. To ensure the detection of low abundance proteins.

TSQ Quantiva is a new generation of triple quadrupole mass spectrometers introduced by Thermo Fisher Scientific at ASMS 2013. TSQ Quantiva uses Active Ion Management (AIM) (Figure 1) technology to optimize ion generation and transfer from ion source to detector, enabling the instrument to quantify the most complex and challenging with the highest sensitivity. Sexual sample. This experiment was designed to examine the sensitivity of TSQ Quantiva to quantify target peptides in complex matrices.

Figure 1. Schematic diagram of active ion management technology (AIM)

Experimental condition

Materials and Methods

PIERCE Peptide Retention Time Calibration Mixture (Cat. No. 88320) contains 15 repetitive peptides with different Hydrophobicity Factor (HF). The 5 ng/μL E.coli digested product was serially diluted to obtain a concentration of 0.0005 fmol/μL, 0.0025 fmol/μL, 0.005 fmol/μL, 0.05 fmol/μL, 0.5 fmol/μL, 5 fmol/μL, 50 fmol. / μL sample. In the experiment, the ion pair was selected by Pinpoint 1.3 software (Fig. 2), and 4 precursor ions were selected for each parent ion, one of which was used as a quantitative ion and three as a qualifier. Finally, 15 target peptides were established, a total of 60 ion pair SRM methods, in which the thickened part was a quantitative ion (Table 1).

Figure 2. Pinpoint 1.3 software for ion pair selection

Table 1. 15 peptide information

High performance liquid chromatography

High Performance Liquid Chromatograph: Thermo Scientific EASY-nLC 1000

Precolumn: Dionex PepMap C18 column (2cm, ID75μm, 3μm)

Analytical column: Dionex PepMap C18 column (15cm, ID75μm, 3μm)

Mobile phase: A, aqueous solution containing 0.1% formic acid; B, acetonitrile solution containing 0.1% formic acid

Gradient: 2%-5% B 2 min, 2%-45% B 42min, 45%-90% B 3 min, 90% B 3min

Flow rate: 300 nL/min

Load capacity: 2 μL

Mass Spectrometry

Mass Spectrometer: Thermo Scientific TSQ Quantiva

Ion source: NanoFlex ion source

Ion mode: positive ion

Spray voltage: 2.0 KV

Capillary temperature: 275 °C

Q1 and Q3 resolution: 0.7 Da;

Cycle time: 2s

Collision energy: The collision energy of the corresponding peptide is calculated by the formula CE=0.034 × m/z + 5.314 of the parent ion, and CE is added or subtracted 2 to further optimize the collision energy.

Experimental result

In this experiment, a full-generation triple quadrupole mass spectrometer TSQ Quantiva was used to quantify 15 heavy-labeled peptides in a complex matrix, and good chromatographic peak shape and mass spectrum response were obtained (Fig. 3). Taking the peptide SSAAPPPPPR* as an example, the detection limit is (LOD) 1 amol and the lower limit of quantitation (LLOQ) is 5 amol (Fig. 4). As shown in the standard curve in Figure 5, the linear correlation coefficient between concentration and peak area is above 0.99 in the concentration range of 5 to 10 fmol, and good repeatability is still obtained at the amol level (Fig. 6). See Table 2 for the standard curve and linear range of the 15 peptides.

Figure 3. Total ion chromatogram (TIC) of 15 peptides (loading 100fmol)

Figure 4. Peptide SSAAPPPPPR* Extraction of different ion fluxes (EIC) m/z493.8-> m/z 335.7

Figure 5. Peptide SSAAPPPPPR* standard curve

Figure 6. Peptide SSAAPPPPPR* repeatability at amol level

Table 2. 15 peptide segments and linear ranges

in conclusion

Experiments show that TSQ Quantiva has outstanding quantitative sensitivity to target peptides in complex matrices thanks to active ion-controlled technology (AIM), greatly improving the ability of the TSQ mass spectrometry platform to quantify target peptides in complex matrices, suitable for complex Analysis of low-abundance proteins in biological samples can be used for confirmation of potential biomarkers.