Peptide and Protein Bioanalysis: Infographic
1
[ PEPTIDE AND PROTEIN BIOANALYSIS ] Aspirin 180 Da Bevacizumab 149 kDa Insulin Glargine 6,063 Da PEPTIDES AND PROTEINS SMALL MOLECULES Multiple precursor charge states dilute signal Increased fragmentation further reduces signal 2+ 3+ 4+ 5+ 1+ In 2013, 7 of the top 10 best-selling prescription drugs were large molecules 2 Humira ® 148 kDa Enbrel ® 150 kDa Remicade ® 149 kDa Lantus ® 6064 Da Rituxan ® 145 kDa Avastin ® 149 kDa Herceptin ® 148 kDa Seretide ® 500 Da Crestor ® 1001 Da Abilify ® 448 Da #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 Increased sensitivity and specificity challenges: Fewer molecules in equal volume of analyte WHY is it harder to quantify large molecules? WHY are biotherapeutics more important than ever? HOW can Waters help? Optimize specificity of sample prep Oasis ® MAX and WCX SPE μElution Plates help achieve high recovery and low limits of detection for a wide range of endogenous, therapeutic and surrogate peptides. Wide mass range, reliable sensitivity T he Xevo ® family of tandem quadrupole mass spectrometers delivers high sensitivity at high and low mass, detecting all fragments without compromise, while sensitivity at high m/z enhances specificity. Improved microfluidics for better sensitivity and reduced sample load T he ionKey/MS™ System integrates the UPLC analytical separation directly into the source of the mass spectrometer, delivering significant increases in sensitivity, solvent savings, and reductions in sample volume. Increasing sensitivity through LC optimization $$ 10x U P T O IMPROVEMENT IN SENSITIVITY More LC and MS parameters to consider reduces efficiency Complex sample prep means possible loss of analyte STEP 1 STEP 2 STEP 3 STEP 4 Year 2014 2010 2005 2000 1995 1990 204 9940 1.2 .80 1.20 1.00 1.00 385 568 2.40 2.00 1.20 1.00 365 240 1.25 1.00 1.25 60 °C 40 °C 400 μL/min 200 μL/min ACN + 0.1% formic acid ACN + 0.1% formic acid + 5% TFE 14237 Temperature Flow rate Mobile phase B composition CAPILLARY VOLTAGE MOBILE PHASE COMPOSITION CHROMATOGRAPHIC PORE SIZE COLUMN TEMP FLOW RATE Analyzing large molecules may be one of the greatest challenges that the bioanalyst faces at the beginning of the 21st century. PEPTIDES AND PROTEINS ARE NOT SMALL MOLECULES. WHY TREAT THEM THE SAME? Biotherapeutics publications 1 Use a generic set of chromatographic conditions and evaluate with the ACQUITY UPLC ® BEH C 18 300 Å, ACQUITY UPLC CSH, and CORTECS ® UPLC C 18 + Columns to identify the best peak and separation and then optimize key parameters, such as column temperature, flow rate, and mobile phase composition. Endogenous peptides and proteins yield high background S E N S I T I V I T Y S P E C I F I C I T Y ©2015 Waters Corporation 720005295EN February 2015 AW-KW Learn more with the Peptide Bioanalysis Solution Guide Search literature code 720004563EN at waters.com 1 Electronic search performed in PubMed.org on January 13, 2015 with keyword “biotherapeutics.” 2 Kollewe, J. (2014, March 27). The world's 10 best-selling prescription drugs. The Guardian. Retrieved Jan. 13, 2015.
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Transcript of Peptide and Protein Bioanalysis: Infographic
[ PEPTIDE AND PROTEIN BIOANALYSIS ]
Aspirin 180 Da Bevacizumab149 kDa
Insulin Glargine6,063 Da
PEPTIDES AND PROTEINSSMALL MOLECULES
Multiple precursorcharge states dilute signal
Increased fragmentationfurther reduces signal
2+
3+ 4+
5+
1+
In 2013, 7 of the top 10 best-selling prescription drugs were large molecules2
Humira®
148 kDa Enbrel®
150 kDa Remicade®
149 kDa Lantus®
6064 Da Rituxan®
145 kDa Avastin®
149 kDa Herceptin®
148 kDa Seretide®
500 Da Crestor®
1001 Da Abilify®
448 Da
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10
Increased sensitivity and specificity challenges:
Fewer molecules inequal volume of analyte
WHY is it harder toquantify large molecules?
WHY are biotherapeuticsmore important than ever?
HOW can Waters help?Optimize specificity of sample prep
Oasis® MAX and WCX SPE µElution Plates help achieve high recovery and low limits of detection for a wide range of endogenous, therapeutic and surrogate peptides.
Wide mass range, reliable sensitivityThe Xevo® family of tandem quadrupole mass spectrometers delivers high sensitivity at high and low mass, detecting all fragments without compromise, while sensitivity at high m/z enhances specificity.
Improved microfluidics for better sensitivity and reduced sample loadThe ionKey/MS™ System integrates the UPLC analytical separation directly into the source of the mass spectrometer, delivering significant increases in sensitivity, solvent savings, and reductions in sample volume.
Increasing sensitivity through LC optimization
$$
10xUP TO
IMPROVEMENT INSENSITIVITY
More LC and MS parameters to consider reduces efficiency
Complex sample prep meanspossible loss of analyte
STEP 1 STEP 2 STEP 3 STEP 4
Year 201420102005200019951990
204
9940
1.2.801.201.00 1.00
385
568
2.402.001.201.00
MRM of 1 Channel ES+
1.32e4Area
365
240
1.251.00 1.25
60 °C
40 °C
400 µL/min
200 µL/minACN + 0.1%formic acid
ACN + 0.1%formic acid + 5% TFE
14237
Temperature Flow rate Mobile phase B composition
CAPILLARY VOLTAGEMOBILE PHASE COMPOSITION
CHROMATOGRAPHIC PORE SIZECOLUMN TEMP
FLOW RATE
Analyzing large molecules may be one of the greatest challenges that the bioanalyst faces at the beginning of the 21st century.
PEPTIDES AND PROTEINS ARE NOT SMALL MOLECULES.
WHY TREAT THEM THE SAME?
Biotherapeuticspublications1
Use a generic set of chromatographic conditions and evaluate with the ACQUITY UPLC® BEH C18 300 Å, ACQUITY UPLC CSH, and CORTECS® UPLC C18+ Columns to identify the best peak and separation and then optimize key parameters, such as column temperature, flow rate, and mobile phase composition.
Endogenous peptides andproteins yield high background
SENSITIVITY
SPECIFICITY
©2015 Waters Corporation 720005295EN February 2015 AW-KW
Learn more with the Peptide Bioanalysis Solution GuideSearch literature code 720004563EN at waters.com
1 Electronic search performed in PubMed.org on January 13, 2015 with keyword “biotherapeutics.”
2 Kollewe, J. (2014, March 27). The world's 10 best-selling prescription drugs. The Guardian. Retrieved Jan. 13, 2015.
