Extended Mass Range Triple Quadrupole for Routine Analysis of High Mass-to-charge Peptide Ions

Application Note

AuthorsLinfeng Wu, Christine A. Miller, Jordy Hsiao, Te-wei Chu, Behrooz Zekavat, and Anabel Fandino

Targeted Proteomics

IntroductionRoutine, high-throughput protein quantitation by LC/Triple Quadrupole analysis uses peptides as surrogates for the corresponding proteins. A critical step to successful analysis is to select unique peptides and suitable MRM transitions for each targeted peptide. Peptides with high m/z precursor or product ions are often not selected for analysis due to instrument mass range limitations. However, to address biological questions these peptides may provide critical information or be the only analytical choice. Some examples where this may arise are membrane proteins with large hydrophobic transmembrane domains, proteins with extensive post-translational modifications, and endogenously produced peptides.

This Application Note demonstrates the performance of the Agilent 6495B Triple Quadrupole for peptides with high m/z product ions, showing the benefits of having a mass range up to m/z 3,000. Tryptically digested monoclonal antibody (mAb) standard and a HeLa cell membrane-enriched extract were used as simple and complex samples, respectively.

2

Experimental MethodProtein/peptide selectionUsing an Agilent 6550 Q-TOF Mass Spectrometer, LC/MS data were acquired in a data-dependent mode on anti-IL8 mAb digest and a HeLa membrane-enriched protein digest. Raw data were searched against the appropriate database using Agilent Spectrum Mill (Rev B.05.00.181 SP1) to identify peptides producing high m/z product ions for testing the performance of the Agilent 6495B Triple Quadrupole Mass Spectrometer.

Table 1. Agilent 6495 Triple Quadrupole MS method.

Parameter SettingIon mode AJS, PositiveGas temperature 150 °CDrying gas flow 15 L/minNebulizer gas 30 psiSheath gas temperature 200 °CSheath gas flow 11 L/minCapillary voltage 3,500 VNozzle voltage 0 VHigh/Low pressure RF voltage 200/110 VDelta EMV 100–200 VQ1 and Q3 resolution Wide/UnitFragmentor 380 VCell accelerator voltage 4 VCycle time 267–369 ms

Table 2. Selected peptides and ions for LC/MS/MS test.

Protein name Peptide Modificationm/z Precursor

m/z MRM Quantifier

m/z MRM Qualifier

Anti-IL8 mAb antibody EEQYN[+1606.6]STYR N-glycan G1F 932.7 204.1 366.1 1,392.6 2,065.8 2,227.9

Sterol O-acyltransferase 1 SSTVPIPTVNQYLYFLFAPTLIYR none 1,402.3 1,215.2 762.5 1,110.1 2,219.2 2,429.3

ATP Synthase subunit d, mitochondrial

NLIPFDQMTIEDLNEAFPETK none 1,233.1 1,063.0 228.1 341.2 474.3 2,125.0

ADP/ATP Translocase 1 YFAGNLASGGAAGATSLC[+57]FVYPLDFAR Carbamidomethylation 1,398.7 718.4 175.1 881.5 1,915.9 2,060.0

LC/MS/MS systemAn Agilent 1290 Infinity UHPLC system was interfaced to a 6495B Triple Quadrupole with a mass range of 3,000 u. The LC/MS experiments were performed using an Agilent Jet Stream (AJS) source, and an Agilent Poroshell 120 EC-C18, 2.1 × 100 mm 2.7 µm column (p/n 695775-902). For the mAb digest, a 3.5 minute MRM method was used and for the HeLa membrane digest, a 21 minute MRM method was used.

For glycopeptide quantification, the mAb digest was diluted in series, and the signal response for each injection level was plotted against the total mAb digest amount on-column.

For the HeLa membrane peptide quantification, the signal response for each product ion was plotted against the relative injection amount on-column.

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Product ions with higher m/z tend to have less background noise or higher signal-to-noise ratios in complex samples.

Figure 1 and Figure 2 show the MS/MS spectrum and the optimized MRM chromatography for two of the peptides. The product ions selected for the MRM method are marked with * in the MS/MS plot.

Glycopeptides produce high m/z product ions, which have large glycan chains. These product ions are informative for interpreting glycan structure.

Results and DiscussionPeptide identification and selectionAnti-IL8 mAb digest and HeLa membrane digest were used for peptide identification and MRM method development with the following results:

One G1F N-glycopeptide from the mAb digest, and three peptides from the HeLa membrane digest with product ions ranging from m/z 204.1 to m/z 2,429.3 were selected for a LC/MS/MS test (Table 2).

A

B×103

200 400 600 800 1,000 1,200 1,400 1,600 1,800 2,000 2,2000

1

2

3

4

5

6

7

8

9

Coun

ts

Mass-to-charge (m/z)

×102

0.9 1.0 1.1Coun

ts

×102

1

1

Coun

ts×102

0

×103

0

×104

0

Coun

tsCo

unts

Coun

ts

Acquisition time (min)

Figure 1. MS/MS spectrum and the optimized MRM chromatography for G1F glycopeptide EEQYN[+1606.6]STYR.

Figure 2. MS/MS spectrum and the optimized MRM chromatography for peptide SSTVPIPTVNQYLYFLFAPTLIYR.

×103

200 600 1,000 1,400 1,800 2,200 2,600 3,0000

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

Coun

ts

Mass-to-charge (m/z)

y15y13y12

y20+2

y18+2

y8

y6

y7

b6

b5b4

b3-H2O

precursor

y18

y20

BA

×101

6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4Coun

ts

×102

1

Coun

ts

×102

05

×102

2

2

×102

Coun

tsCo

unts

Coun

ts

Acquisition time (min)

4

Glycopeptide quantificationAn 1290 Infinity UHPLC system coupled with Agilent Jet Stream Technology, and a 6495B Triple Quadrupole LC/MS system with a mass range of 3,000 u were used for glycopeptide quantitation with the following results (Figure 3):

• Excellent precision and accuracy at all levels including injection amount as low as 0.0197 ng (130 amol) of total mAb digest on-column (18.5 % RSD, 97.5 % accuracy). This target peptide is estimated to represent about 10 % of the total protein so LOQ for this glycopeptide is estimated at about 13 amol on-column

• Reproducible responses for product ions with extended mass range (m/z 204.1 ~ m/z 2,227.9)

• Excellent retention time (RT) reproducibility (RSD% = 0.47 % for n = 90)

• Excellent linearity at low injection levels with small fold changes (0.0197 ng ~ 19.7 ng, R2 = 0.998)

EEQYN(+1606.586706)STYR

Concentration (ng of mAb digest on-column)-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

-0.10

0.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.9

2

y = 943.671754*x – 5.093398R2 = 0.99814103

Resp

onse

0 0.04 0.08 0.12 0.16 0.20 0.24 0.28-0.20.20.61.01.41.82.22.63.0

Resp

onse

Amount of total mAb digest on-column (ng)

%RSD(n = 10) % Accuracy

0.0197 18.5 97.5

0.0394 18.3 104.2

0.0591 12.5 108.3

0.0985 9.3 102.4

0.197 13.0 95.6

0.394 5.9 84.8

0.985 5.7 102.1

1.97 1.6 105.5

19.7 2.9 99.7

Blank

0.0197 ng

0.0394 ng

0.0591 ng

0.0098 ng

Zoom

Zoom

Resp

onse

Resp

onse

Replicate injection

932.7 & 204.1932.7 & 2,227.9932.7 & 2,065.8932.7 & 1,392.6932.7 & 366.1

A

C

D

B

×102

×104

×102

0.90

1 2 3 4 5 6 7 8 9 10Replicate injection

1 2 3 4 5 6 7 8 9 10

0.95 1.00 1.05 1.10

Coun

ts

2.6

0 0200400600800

1,0001,2001,400

2,0004,0006,0008,000

10,00012,00014,00016,00018,00020,000

×102

Coun

ts

2.6

×102

Coun

ts

2.5

×102Co

unts

2.462.48

×102

Coun

ts

2.46

2.48

Acquisition time (min)

Figure 3. Quantification for G1F glycopeptide EEQYN[+1606.6]STYR.

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• Good linearity was observed (R2 ranges from 0.989 to 0.997).

• Reproducible responses for product ions with extended mass range (up to m/z 2429.3) were observed (right plots).

• The quantifier response for each of these three peptides were plotted against the relative injection amount on-column (left plots) as the stoichiometry of these peptides were unknown in the sample.

HeLa membrane peptide quantificationThree peptides from the HeLa membrane-enriched sample digest were quantified following the same protocol as the glycopeptide to test high m/z product ion behavior (Figure 4).

Figure 4. Quantification for peptides from a HeLa membrane digest.

YFAGNLASGGAAGATSLCFVYPLDFAR

Concentration (relative amount on-column)0 4 8 12 16 20 24 28 32 36 40 44 48 52

Concentration (relative amount on-column)0 4 8 12 16 20 24 28 32 36 40 44 48 52

Concentration (relative amount on-column)0 4 8 12 16 20 24 28 32 36 40 44 48 52

-0.10

0.10.20.30.40.50.60.70.80.91.0 y = 202.884088*x – 54.476396

R2 = 0.99576872

NLIPFDQMTIEDLNEAFPETK

-0.10

0.10.20.30.40.50.60.70.80.91.01.1

y = 205.558590*x – 13.485689R2 = 0.99704134

SSTVPIPTVNQYLYFLFAPTLIYR

-0.10

0.10.20.30.40.50.60.70.80.91.01.11.21.31.41.5

y = 29.206852*x – 10.518556R2 = 0.98908869

×104

×104

×103

Resp

onse

Resp

onse

Resp

onse

Resp

onse

Replicate injection1 2 3 4 5

0

2,000

4,000

6,000

8,000

10,000

12,000Re

spon

se

Replicate injection1 2 3 4 5

0

2,000

4,000

6,000

8,000

10,000

12,000

Resp

onse

Replicate injection1 2 3 4 5

0200400600800

1,0001,2001,4001,600

1,398.7 & 718.41,398.7 & 2,060.01,398.7 & 1,915.91,398.7 & 881.51,398.7 & 175.1

1,233.1 & 1,063.01,233.1 & 2,125.01,233.1 & 474.31,233.1 & 341.21,233.1 & 228.1

1,402.3 & 1,215.21,402.3 & 2,429.31,402.3 & 2,219.21,402.3 & 1,110.11,402.3 & 762.5

000000000

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For Research Use Only. Not for use in diagnostic procedures.

This information is subject to change without notice.

© Agilent Technologies, Inc., 2016, 2018 Published in the USA, March 16, 2018 5991-7298EN

ConclusionsThe Agilent 6495B Triple Quadrupole LC/MS system has a mass range of 3,000 u, which is useful for detecting high m/z peptide ions. One benefit of using high m/z precursor or product ions, particularly in a complex digest, is these ions have less background noise compared to those with low m/z. Some high m/z product ions may provide important biological information such as location and size of post-translational modifications, for example glycosylation.

This application notes demonstrates the excellent reproducibility for MRM transitions with high m/z product ions achieved on the Agilent 6495B Triple Quadrupole LC/MS system. The work shown here uses Agilent Jet Stream Technology at standard flow rates and resulted in outstanding precision and accuracy for both glycopeptide and HeLa membrane peptide quantification.