jetstream technology: mass- or concentration sensitive
TRANSCRIPT
ASMS template for 2004Introduction
Conclusions
NanoLC-MS with 100 um ID columns are often used in Proteomics, due to the high sensitivity of nanospray and optimal use of the small sample amount. Sample are loaded using up-front enrichment columns which allow loadability of 1-2 µg. of protein digest. More sample may be available but cannot be used due to breakthrough and overloading of the column. Ease of use and robust operation, also, remain a challenge. Many researchers are therefore trying capillary columns (300 um ID) which are operated using direct injection, It circumvents the problems with enrichment and according to conventional rules for electrospray ionization with concentration sensitive response, allows the handling of sample in the lowest possible volume and thus maximum sensitivity. In recent years, UHPLC has improved speed and resolution in chromatography using sub-2-micron and core-shell particle technology. A increase of 12x in loadability would be possible over capillary columns. Can UHPLC/MS using 2.1 mm columns combined with modern ionization sources challenge nano-LC-MS performance in Proteomics?
Discovery Proteomics applications were investigated using 2.1 mm. ID UHPLC columns on a 6550 QTOF (Agilent Jetstream and iFunnel)
Jetstream Proteomics is a unique solution that uses 2.1 mm. ID UHPLC Columns together with Jet Stream and ion funnel technology. It is used for both discovery and targeted experiments, enables near nanoflow sensitivity for proteomics and is intended for scientists who are not sample limited (plasma, cell cultures, plants, food, etc.) It offers the ease-of-use, robustness and reproducibility associated with standard flow separations. It features faster analysis times for complex samples and faster re-equilibration times. For targeted proteomics, superior retention-time reproducibility and thus narrower MRM retention-time windows. For discovery proteomics equivalent protein identifications as nanoflow using 20-30 µg of sample.
Quantitative Proteomics Discovery Proteomics
Jetstream Technology: Mass- or concentration sensitive? The impact for LC-MS of complex samples Tom van de Goor1, Stephan Buckenmaier1, Christine Miller2 and Monika Dittmann1,Agilent Technologies, Waldbronn, Germany1 and Santa Clara, CA, USA2 HPLC 2015 Poster PSA-LCMS-33
Subhead (34 bold)
Details here.
Ionization with Agilent Jetstream Technology New ionization approaches such as Jetstream technology have enabled more efficient ionization at standard UHPLC flow rates. Super-heated sheath gas helps drying the solvent droplets, allowing more ions to be formed and focusing the ion plume for better sampling into the MS inlet. For peptides, AJS leads to a typical increase of 3x in sensitivity.
Figure 1: The Agilent Jetstream Technology vs. Standard ESI
Sampling with Agilent iFunnel Technology The inlet capillary on the mass spectrometer has been replaced by a hexabore sampling inlet, which allows more gas and thus more ions to be transferred. A dual ion funnel helps removal of neutrals and guides the ions of interest into the low pressure region and leads to 10x increase in sensitivity.
Figure 3: The Agilent iFunnel Technology and its sensitivity advantage.
0.2
0.4
0.6
0.8
1.0
Acquisition Time (min) 3 3.5 4 4.5 5 5.5 6 6.5 7
AJS normalized response
ESI relative response
Figure 2: Normalized response of peptides using AJS vs. Standard ESI
Concentration or Mass sensitive response A mixture 15 pesticides and 7 peptides where analyzed using comparable gradient conditions on 4 different ID columns (0.3, 0.5, 1.0 and 2.1 mm ID). In all cases the columns were packed with the same 3.5 μm fully porous particles and the column length was the same at 150 mm. and gradients ran from 5-50% H2O/ACN with 0.1% formic acid. Sample concentration was 100 ng/μL for UV measurements. The sample was diluted 100x for ESI and 500x for JetStream. In all cases 0.5 μL was injected. Results below show either UV at 254 nm or the TIC for MS. Agilent Jetstream shows mass sensitive response up to 2.1 mm. ID columns.
UV (254 nm) Concentration sensitive
Conventional ESI Mixed response
ID=0.5 mm, F=20 μL/min
ID=1.0 mm, F=100 μL/min
ID=2.1 mm, F=430 μL/min
ID=2.1 mm, F=430 μL/min – Zoom-In
ID=0.3 mm, F=9 μL/min
ID=1.0 mm, F=100 μL/min
ID=1.0 mm, F=100 μL/min
ID=0.5 mm, F=20 μL/min
ID=2.1 mm, F=430 μL/min
ID=2.1 mm, F=430 μL/min
Figure 4: Concentration sensitivity (UV) vs. Mass sensitivity (AJS)
Quantitative Proteomics applications were investigated using 2.1 mm. ID UHPLC columns on a 6495 QQQ (Agilent Jetstream and iFunnel)
Peptide Quantitation in Plasma For complex biological matrices such as plasma, the improved chromatographic performance of standard UHPLC/MS allows short analysis time, high throughput and robust assays. The outstanding retention time stability, allows creation of tight windows for scheduled MRM transitions which gives more sensitivity by spending more time on each transition. During a 3.5 week long measurement series of plasma digests, retention time RSD was less than 1.5% for 40 peptides monitored using a QC kit of MRM Proteomics Inc.
6x10
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0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1 1
Counts vs. Acquisition Time (min)
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Figure 5: Overlay of 238 transitions of 40 peptides (MRM Proteomics QC) LC Conditions: INFINITY BINARY SYSTEM
Column AdvanceBio Peptide Mapping, 2.1 x 100 mm, 2.7 µm (pn 655750-902)
Column temperature 50 ºC
Injection volume 20 µL
Autosampler temp 4 ºC
Needle wash 10 seconds in wash port (50:50 water:methanol with 0.1% formic acid)
Mobile phase A = 0.1% formic acid in water B = 0.1% formic acid in 90% acetonitrile in water
Flow rate 0.40 mL/min
Stop time 27.5 min
Post time 4.0 min
AGILENT 6495 QQQ Setting
Ion mode AJS, Positive
Gas temperature 150 ºC
Nebulizer gas 30 psi
Sheath gas temperature 250ºC
Capillary voltage 3500 V
Nozzle voltage 500 V
Delta DMV 400 V
Fragmentor 380 V
Cycle time 240 msec
Min/Max Dwell 2.89/38.63
Peptide LLOQ in an Enolase Digest The LLOQ for a synthetic peptide from Human Serum Albumin (LVNEVTEFAK), spiked in an Enolase tryptic digest was determined to be 5 amol on-column with a six-orders-of-magnitude dynamic range. This demonstrates that sensitivity is not compromised when moving to 2.1 mm. ID columns for routine quantitative Proteomics. Reproducibility at the lower limit of quantitation was 14% (n=10) and accuracy was 109.8%.
Figure 6: LLOD and LLOQ of synthetic peptide in an Enolase tryptic digest
LC Conditions
AGILENT INFINITY BINARY SYSTEM
Column Eclipse Plus EC-C18 RRHD 2.1 x 50 mm, 1.8 um column (pn 857750-902)
Column temperature 35 ºC
Injection volume 1 µL
Autosampler temp 4 ºC
Needle wash 10 seconds in wash port (50:50 water:methanol with 0.1% formic acid)
Mobile phase A = 0.1% formic acid in water B = 0.1% formic acid in 90% acetonitrile in water
Flow rate 0.60 mL/min
Stop time 5.0 min Post time off
AGILENT 6495 QQQ Setting
Ion mode AJS, Positive Gas temperature 150 ºC Drying gas flow 15 L/min Nebulizer gas 30 psi Sheath gas temperature 200ºC
Sheath gas flow 11 L/min Capillary voltage 3500 V Nozzle voltage 0 V High/Low Pressure RF voltage
200/110 V
Fragmentor 380 V Cell Accelerator Voltage 4 V
Cycle time 310.5 ms/cycle Total MRMs 3 Transitions (Collision Energy)
575.3111 937.4625 (15.9 V)
References
Protein Identification in an E. Coli digest Data dependent acquisition using nano-LC/MS has long been the preferred technique due to the wide dynamic range and sensitivity requirements. To improve depth of coverage, columns would typically be overloaded, which led to shifts in chromatographic retention and displacement effects. A Escherichia coli (E. coli) tryptic digest was used to demonstrate the effect of gradient length and sample loading on protein and peptide identification using 2.1 mm ID UHPLC columns.
Figure 7: Proteins and peptides identified in an E.Coli tryptic digest as a function of the gradient length and sample amount loaded on column
LC Conditions INFINITY BINARY SYSTEM Column AdvanceBio Peptide Mapping, 2.1 x 250 mm, 2.7 µm (pn 651750-902)
Column temperature 50 ºC
Injection volume 20 µL
Autosampler temp 4 ºC
Needle wash 10 seconds in wash port (50:50 water:methanol with 0.1% formic acid)
Mobile phase A = 0.1% formic acid in water B = 0.1% formic acid in 90% acetonitrile in water
Flow rate 0.40 mL/min
B (%) 3 40 70 70 3
Stop time 60.0 min. 90.0 min. 120.0 min 150.0 min.
Post time 5.0 min
AGILENT 6550 QTOF Setting
Ion Source/Mode Agilent Jet Stream, Positive
Gas temperature 250 ºC Drying gas flow 14 L/min Nebulizer gas 35 psi Sheath gas temperature 250ºC
Sheath gas flow 11 L/min Capillary voltage 3500 V Nozzle voltage 0 V Fragmentor 360 V
Reference mass 322.048121 and 1221.990637
Longer gradient times increase the number of protein identifications, although the benefit is not as dramatic as observed in nanoflow LC/MS. The amount of MS/MS spectra that could be validated was greater than 60%, which support the use of shorter methods. Sample loading could be increased by a factor of 10 over nano and capillary columns without overload to 15 µg on-column. For all database searching Spectrum Mill SW was used with a false discovery rate (FDR) filter set at 1.2%.
Protein Identification in a MDA-MB-231 cell lysate An even more complex sample of 25ug (2ug/uL in 0.1% formic acid) Human breast cancer cell line was analyzed in triplicate in order to reveal as many peptides and proteins as possible. The identification of 32,446 peptides, representing 5,905 unique proteins demonstrates the power of using standard UHPLC columns.
23016 22863 22669 32446
4,875 4,824 4,946 5,905
Unique peptides: 32,446 Unique Proteins: 5,905
Figure 8: Protein Identification in a human breast cancer cell line. LC Conditions INFINITY BINARY SYSTEM Column AdvanceBio Peptide Mapping, 2.1 x 250 mm, 2.7 µm (pn 651750-902)
Column temperature 50 ºC
Injection volume 12.5 µL
Mobile phase A = 0.1% formic acid in water B = 0.1% formic acid in 90% acetonitrile in water
Flow rate 0.20 mL/min
B (%) 5 25 40 90 5
Stop time 140.0 min
2. iFunnel Technology for Enhanced sensitivity in Tandem LC/MS: Publication Number 5990-5891EN.
3. Buckenmaier et al., Instrument contributions to resolution and sensitivity in ultra high performance liquid chromatography using small bore columns, J. Chrom. A 2015, 1377 pp. 64-74.
4. Jetstream Proteomics for sensitive and robust standard flow LC/MS: Publication Number 5991-5687EN.
Slide Number 1
Conclusions
NanoLC-MS with 100 um ID columns are often used in Proteomics, due to the high sensitivity of nanospray and optimal use of the small sample amount. Sample are loaded using up-front enrichment columns which allow loadability of 1-2 µg. of protein digest. More sample may be available but cannot be used due to breakthrough and overloading of the column. Ease of use and robust operation, also, remain a challenge. Many researchers are therefore trying capillary columns (300 um ID) which are operated using direct injection, It circumvents the problems with enrichment and according to conventional rules for electrospray ionization with concentration sensitive response, allows the handling of sample in the lowest possible volume and thus maximum sensitivity. In recent years, UHPLC has improved speed and resolution in chromatography using sub-2-micron and core-shell particle technology. A increase of 12x in loadability would be possible over capillary columns. Can UHPLC/MS using 2.1 mm columns combined with modern ionization sources challenge nano-LC-MS performance in Proteomics?
Discovery Proteomics applications were investigated using 2.1 mm. ID UHPLC columns on a 6550 QTOF (Agilent Jetstream and iFunnel)
Jetstream Proteomics is a unique solution that uses 2.1 mm. ID UHPLC Columns together with Jet Stream and ion funnel technology. It is used for both discovery and targeted experiments, enables near nanoflow sensitivity for proteomics and is intended for scientists who are not sample limited (plasma, cell cultures, plants, food, etc.) It offers the ease-of-use, robustness and reproducibility associated with standard flow separations. It features faster analysis times for complex samples and faster re-equilibration times. For targeted proteomics, superior retention-time reproducibility and thus narrower MRM retention-time windows. For discovery proteomics equivalent protein identifications as nanoflow using 20-30 µg of sample.
Quantitative Proteomics Discovery Proteomics
Jetstream Technology: Mass- or concentration sensitive? The impact for LC-MS of complex samples Tom van de Goor1, Stephan Buckenmaier1, Christine Miller2 and Monika Dittmann1,Agilent Technologies, Waldbronn, Germany1 and Santa Clara, CA, USA2 HPLC 2015 Poster PSA-LCMS-33
Subhead (34 bold)
Details here.
Ionization with Agilent Jetstream Technology New ionization approaches such as Jetstream technology have enabled more efficient ionization at standard UHPLC flow rates. Super-heated sheath gas helps drying the solvent droplets, allowing more ions to be formed and focusing the ion plume for better sampling into the MS inlet. For peptides, AJS leads to a typical increase of 3x in sensitivity.
Figure 1: The Agilent Jetstream Technology vs. Standard ESI
Sampling with Agilent iFunnel Technology The inlet capillary on the mass spectrometer has been replaced by a hexabore sampling inlet, which allows more gas and thus more ions to be transferred. A dual ion funnel helps removal of neutrals and guides the ions of interest into the low pressure region and leads to 10x increase in sensitivity.
Figure 3: The Agilent iFunnel Technology and its sensitivity advantage.
0.2
0.4
0.6
0.8
1.0
Acquisition Time (min) 3 3.5 4 4.5 5 5.5 6 6.5 7
AJS normalized response
ESI relative response
Figure 2: Normalized response of peptides using AJS vs. Standard ESI
Concentration or Mass sensitive response A mixture 15 pesticides and 7 peptides where analyzed using comparable gradient conditions on 4 different ID columns (0.3, 0.5, 1.0 and 2.1 mm ID). In all cases the columns were packed with the same 3.5 μm fully porous particles and the column length was the same at 150 mm. and gradients ran from 5-50% H2O/ACN with 0.1% formic acid. Sample concentration was 100 ng/μL for UV measurements. The sample was diluted 100x for ESI and 500x for JetStream. In all cases 0.5 μL was injected. Results below show either UV at 254 nm or the TIC for MS. Agilent Jetstream shows mass sensitive response up to 2.1 mm. ID columns.
UV (254 nm) Concentration sensitive
Conventional ESI Mixed response
ID=0.5 mm, F=20 μL/min
ID=1.0 mm, F=100 μL/min
ID=2.1 mm, F=430 μL/min
ID=2.1 mm, F=430 μL/min – Zoom-In
ID=0.3 mm, F=9 μL/min
ID=1.0 mm, F=100 μL/min
ID=1.0 mm, F=100 μL/min
ID=0.5 mm, F=20 μL/min
ID=2.1 mm, F=430 μL/min
ID=2.1 mm, F=430 μL/min
Figure 4: Concentration sensitivity (UV) vs. Mass sensitivity (AJS)
Quantitative Proteomics applications were investigated using 2.1 mm. ID UHPLC columns on a 6495 QQQ (Agilent Jetstream and iFunnel)
Peptide Quantitation in Plasma For complex biological matrices such as plasma, the improved chromatographic performance of standard UHPLC/MS allows short analysis time, high throughput and robust assays. The outstanding retention time stability, allows creation of tight windows for scheduled MRM transitions which gives more sensitivity by spending more time on each transition. During a 3.5 week long measurement series of plasma digests, retention time RSD was less than 1.5% for 40 peptides monitored using a QC kit of MRM Proteomics Inc.
6x10
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1 1
Counts vs. Acquisition Time (min)
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Figure 5: Overlay of 238 transitions of 40 peptides (MRM Proteomics QC) LC Conditions: INFINITY BINARY SYSTEM
Column AdvanceBio Peptide Mapping, 2.1 x 100 mm, 2.7 µm (pn 655750-902)
Column temperature 50 ºC
Injection volume 20 µL
Autosampler temp 4 ºC
Needle wash 10 seconds in wash port (50:50 water:methanol with 0.1% formic acid)
Mobile phase A = 0.1% formic acid in water B = 0.1% formic acid in 90% acetonitrile in water
Flow rate 0.40 mL/min
Stop time 27.5 min
Post time 4.0 min
AGILENT 6495 QQQ Setting
Ion mode AJS, Positive
Gas temperature 150 ºC
Nebulizer gas 30 psi
Sheath gas temperature 250ºC
Capillary voltage 3500 V
Nozzle voltage 500 V
Delta DMV 400 V
Fragmentor 380 V
Cycle time 240 msec
Min/Max Dwell 2.89/38.63
Peptide LLOQ in an Enolase Digest The LLOQ for a synthetic peptide from Human Serum Albumin (LVNEVTEFAK), spiked in an Enolase tryptic digest was determined to be 5 amol on-column with a six-orders-of-magnitude dynamic range. This demonstrates that sensitivity is not compromised when moving to 2.1 mm. ID columns for routine quantitative Proteomics. Reproducibility at the lower limit of quantitation was 14% (n=10) and accuracy was 109.8%.
Figure 6: LLOD and LLOQ of synthetic peptide in an Enolase tryptic digest
LC Conditions
AGILENT INFINITY BINARY SYSTEM
Column Eclipse Plus EC-C18 RRHD 2.1 x 50 mm, 1.8 um column (pn 857750-902)
Column temperature 35 ºC
Injection volume 1 µL
Autosampler temp 4 ºC
Needle wash 10 seconds in wash port (50:50 water:methanol with 0.1% formic acid)
Mobile phase A = 0.1% formic acid in water B = 0.1% formic acid in 90% acetonitrile in water
Flow rate 0.60 mL/min
Stop time 5.0 min Post time off
AGILENT 6495 QQQ Setting
Ion mode AJS, Positive Gas temperature 150 ºC Drying gas flow 15 L/min Nebulizer gas 30 psi Sheath gas temperature 200ºC
Sheath gas flow 11 L/min Capillary voltage 3500 V Nozzle voltage 0 V High/Low Pressure RF voltage
200/110 V
Fragmentor 380 V Cell Accelerator Voltage 4 V
Cycle time 310.5 ms/cycle Total MRMs 3 Transitions (Collision Energy)
575.3111 937.4625 (15.9 V)
References
Protein Identification in an E. Coli digest Data dependent acquisition using nano-LC/MS has long been the preferred technique due to the wide dynamic range and sensitivity requirements. To improve depth of coverage, columns would typically be overloaded, which led to shifts in chromatographic retention and displacement effects. A Escherichia coli (E. coli) tryptic digest was used to demonstrate the effect of gradient length and sample loading on protein and peptide identification using 2.1 mm ID UHPLC columns.
Figure 7: Proteins and peptides identified in an E.Coli tryptic digest as a function of the gradient length and sample amount loaded on column
LC Conditions INFINITY BINARY SYSTEM Column AdvanceBio Peptide Mapping, 2.1 x 250 mm, 2.7 µm (pn 651750-902)
Column temperature 50 ºC
Injection volume 20 µL
Autosampler temp 4 ºC
Needle wash 10 seconds in wash port (50:50 water:methanol with 0.1% formic acid)
Mobile phase A = 0.1% formic acid in water B = 0.1% formic acid in 90% acetonitrile in water
Flow rate 0.40 mL/min
B (%) 3 40 70 70 3
Stop time 60.0 min. 90.0 min. 120.0 min 150.0 min.
Post time 5.0 min
AGILENT 6550 QTOF Setting
Ion Source/Mode Agilent Jet Stream, Positive
Gas temperature 250 ºC Drying gas flow 14 L/min Nebulizer gas 35 psi Sheath gas temperature 250ºC
Sheath gas flow 11 L/min Capillary voltage 3500 V Nozzle voltage 0 V Fragmentor 360 V
Reference mass 322.048121 and 1221.990637
Longer gradient times increase the number of protein identifications, although the benefit is not as dramatic as observed in nanoflow LC/MS. The amount of MS/MS spectra that could be validated was greater than 60%, which support the use of shorter methods. Sample loading could be increased by a factor of 10 over nano and capillary columns without overload to 15 µg on-column. For all database searching Spectrum Mill SW was used with a false discovery rate (FDR) filter set at 1.2%.
Protein Identification in a MDA-MB-231 cell lysate An even more complex sample of 25ug (2ug/uL in 0.1% formic acid) Human breast cancer cell line was analyzed in triplicate in order to reveal as many peptides and proteins as possible. The identification of 32,446 peptides, representing 5,905 unique proteins demonstrates the power of using standard UHPLC columns.
23016 22863 22669 32446
4,875 4,824 4,946 5,905
Unique peptides: 32,446 Unique Proteins: 5,905
Figure 8: Protein Identification in a human breast cancer cell line. LC Conditions INFINITY BINARY SYSTEM Column AdvanceBio Peptide Mapping, 2.1 x 250 mm, 2.7 µm (pn 651750-902)
Column temperature 50 ºC
Injection volume 12.5 µL
Mobile phase A = 0.1% formic acid in water B = 0.1% formic acid in 90% acetonitrile in water
Flow rate 0.20 mL/min
B (%) 5 25 40 90 5
Stop time 140.0 min
2. iFunnel Technology for Enhanced sensitivity in Tandem LC/MS: Publication Number 5990-5891EN.
3. Buckenmaier et al., Instrument contributions to resolution and sensitivity in ultra high performance liquid chromatography using small bore columns, J. Chrom. A 2015, 1377 pp. 64-74.
4. Jetstream Proteomics for sensitive and robust standard flow LC/MS: Publication Number 5991-5687EN.
Slide Number 1