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The world leader in serving science
ZipChip™ System for Selected Thermo Scientific™ Mass Spectrometers-- for CE/ESI-MS Analyses
2
Content
Resources and Ordering Information
ZipChip System Introduction
ZipChip Separation Mechanism
Biopharmaceutical Application Examples
Metabolomics Application and System Test
3
• The ZipChip system uses integrated
microfluidic technology to prepare,
separate samples by capillary electrophoresis (CE), and then
electrospray (ESI) analytes directly into
a mass spectrometer (MS)
• It is composed of the ZipChip interface
and the microfluidic chip
• ZipChip Interface directly mounts onto
the front end of a mass spectrometer
• The CE separation and ESI occur on the microfluidic chip
• ZipChip system is compatible with a
broad range of biomatrices such as growth media, cell lysates, blood,
plasma, and urine
• The ZipChip system uses integrated
microfluidic technology to prepare,
separate samples by capillary electrophoresis (CE), and then
electrospray (ESI) analytes directly into
a mass spectrometer (MS)
• It is composed of the ZipChip interface
and the microfluidic chip
• ZipChip Interface directly mounts onto
the front end of a mass spectrometer
• The CE separation and ESI occur on the microfluidic chip
• ZipChip system is compatible with a
broad range of biomatrices such as growth media, cell lysates, blood,
plasma, and urine
What is the ZipChip System?
ZipChip HS ZipChip HR
Separation channel length (cm) 10 22
Flowrate (nL/min) 150 150
Maximum # of injections per chip 125 125
On Chip De-salting capability Yes Yes
Integrated ESI Emitter Yes Yes
EEPROMS (recognize chip type and track usage) Yes Yes
Recommended use Small molecules or simple sample mixture Big molecules or complex sample mixture
Typical analysis time Up to 3 min Up to 15 min
4
Why the ZipChip System?
• Capillary Electrophoresis (CE) has unique advantages for certain analyses where traditional LC has its limitation
Separation of charge variants of intact antibodies/proteins
Effective separation of monosaccharides, glycans, and glycopeptides in a MS compatible condition
Separation of very hydrophilic molecules Separation of amino acid isomers
Analyses of samples in high salt condition
• Other CE instruments that can be coupled with MS are bulky with cumbersome operations and take longer analysis time
• They are not specifically designed to be coupled with MS therefore requires additional interface
• There is a lack of convenient CE/ESI device that offers fast and good CE separation for mass spectrometry analysis
• ZipChip is the ONLY commercially available portable and integrated CE/ESI source designed for mass spectrometers
Fast CE separation • Nano Spray Sensitivity • HRAM Mass Spectrometry
5
Unique Values and Common Applications
• Intact mAb and ADC/biotherapeuticscharacterization
Native condition Denatured condition
• mAb subunit analyses
• Glycomics and glycoproteomics
• Peptide mapping
• Metabolomics
• The only commercially available integrated and portable CE/ESI interface for MS
• Offers extremely rapid CE separations, nano-spray level sensitivity, and HRAM mass spectrometry in one platform
• Requires minimal sample preparation with on-chip desalting capability
• Consumes only picograms to nanograms of sample per analysis
Common Applications (not a full list)Unique Values
6
Comprehensive Portfolio
ZipChip Interface ZipChip Autosampler ZipChip Assay KitsZipChips
• Compatible with all Thermo
ScientificTM Exactive, Q
Exactive Orbitrap MS, and
LTQ Orbitrap MS instruments
• Data collection, processing
and reporting through
Thermo ScientificTM
Xcalibur and BioPharma
Finder software
• Two versions: Autosampler
operation version and manual operation version
• Disposable chips
• Good for up to 125
injections
• Two types: HR chip and HS chip
• Each chip is a single piece
of glass similar as a
microscope slide, housed in PEEK polymer
• Microfluidic channel and
electrospray is integrated
into both types of chips• 5 chips per pack
• 3 types of pre-made assay kits
are designed for intact antibody,
peptides, and metabolites
analyses, respectively• Each box of assay kit contains
5 bottles of BGE and 5 bottles
of sample diluents.
• Individual bottles can be directly loaded into ZipChip
autosampler
• Each individual bottle contains
enough volume for 100 runs• Each box is good for 500 runs
• Required to have for
ZipChip Interface
autosampler version
• Fully automated and controlled by the ZipChip
software
• Conduct chip priming,
sample and BGE loading, and chip flushing
• Compatible with both 48-
vial plate and 96-well plate
DryDock
• A simply drying
accessary
• No electrical
component• Utilize the MS gas to
push air through the
ZipChip channels
removing residual fluid
• Dry 1 chip each time
7
ZipChip-MS Analysis Workflow
Place ZipChip and prepare the system
3
Simple Sample Prep
2
Set up sequence and collect CE-MS data
4
Select proper assay kit and ZipChip for your experiments
1
8
Content
Resources and Ordering Information
ZipChip System Introduction
ZipChip Separation Mechanism
Biopharmaceutical Application Examples
Metabolomics Application and System Test
9
Anatomy of ZipChip
MS Inlet
Glass microfluidic chip in PEEK housing
+HV1
+HV2
Well 1Waste
Well 2BGE
Well 3Sample
Well 4BGE
Well 2BGE
Well 3Sample
Well 1Waste
Well 4BGE
10
+HV1
+HV2
Well 1Waste
Well 2BGE
Well 3Sample
Well 4
BGE
Sample Injection
10 µM rhodamine-6G in BGE + 100 mM ammonium acetate
11
Sample Separation
• High voltage applied to Wells 2 and 4
• HV1 and HV2 determine field strength
• Field strength drives the ZipChipseparation
+HV1
+HV2
Well 1Waste
Well 2BGE
Well 3Sample
Well 4BGE
��� ��
6��
q - chargeη - viscosity - hydrodynamic radius
�� !" #$% &'$ℎ �)*1 , )*2
.ℎ&& ! / &'$ℎ
For ZipChip analysis analytes must be positively charged in solution
Cations: Migrate to ESI emitter and are electrosprayed
Anions: Migrate to Well 2
Neutrals: Do not migrate. Pushed to waste by EOF
--
++
Residual EOF++
++++++
+HV1
ESI Emitter
Well 1
(Waste)
Well 3
(Sample)
Well 2 (BGE)
--
------
(Electro-osmotic Flow)
12
Content
Resources and Ordering Information
ZipChip System Introduction
ZipChip Separation Mechanism
Biopharmaceutical Application Examples
Metabolomics Application and System Test
13
The ZipChip System with Thermo ScientificTM Q ExactiveTM BioPharma Platform
BioPharma Option is available for Q Exactive Plus, Q Exactive HF and Q Exactive HF-X mass spectrometers
14
The ZipChip system coupled with the
Q Exactive BioPharma platform can
quickly analyze intact mAbs in native, partially denatured, and fully
denatured conditions to support
biotherapeutics characterizations
under a diverse range of conditions
• CE/ESI-MS analysis can be
completed within 3 minutes
• High resolution accurate mass spectra in intact native, partially
denatured, and fully denatured
states on the Q Exactive
Plus/HF/HF-X MS with BioPharmaoption are confidently achieved
• Sample consumption can be as low
as pico grams to nano grams
• Major glycoforms are identified by
BioPharma Finder software
G0/G0F
G0F/G0F
G0F/G1F
(G1F/G1F) or (G0F/G2F)
G1F/G2F
1.0 ppm
3.9 ppm
1.0 ppm
7.5 ppm
2.4 ppm
0
20
40
60
80
100 148056.78
148218.15
148379.38
147910.25 148542.25
147800 148200 148600Mass
Rela
tive
Inte
nsity
1 2 3
Time (min)
0
100
0
100
Rela
tive A
bundance
0
1000.96
2.35
2.51
a
b
c
Fully denatured
Partially denatured
z=26
z=30
z=49
2000 4000 6000 8000m/z0
20
40
60
80
1000
20
40
60
80
100
Re
lative
Ab
un
dan
ce
0
20
40
60
80
1005695.4
5923.2
5484.6
5288.7 6183.7
3022.6
3085.5
3151.2
3444.2
4936.2
4777.1
5106.4
4627.83897.2 5288.8
3797.4
Native
5660 5700 5740m/z
5695.4
5701.7
5707.7
5689.8 5714.0
a
b
c
1 ng of sample
injected
160 pg of sample
injected
160 pg of sample
injected
Full MS spectra from Q Exactive HF-X MS with BioPharma Option
Electropherograms of intact Trastuzumab Deconvoluted spectrum
z=38
Intact mAb - Trastuzumab Analysis in HMR mode
MS data was acquired on a Q Exactive HF-X MS
with BioPharma OptionCE separation was achieved on ZipChip HR
with 10mM Ammonium Acetate and 10% Isopropanol (native),
Intact Antibody Assay kit (partially denatured), or Peptides Assay Kit (fully denatured)
Resolution: 30K
15
Electropherogram of intact infliximab:
0 2 4 6 8Time (min)0
20
40
60
80
100
Rela
tive A
bundance
2.6
4.53.5
0 Lys2 Lys1 Lys
5700 5720 5740m/z
0
50
1000
50
100
Rela
tive A
bundance
0
50
1005722.8
5717.9
5713.0
zoom
z=26Full MS spectra from Q Exactive HF-X MS with BioPharma Option
5000 6000m/z
0
50
1000
50
100
Rela
tive A
bundance
0
50
1005722.85510.8
5314.1 5951.7
5717.95506.2 5946.6
5309.5
5713.05941.45501.4
5305.0
z=26
z=25
z=27
z=28 RT 2.6 min
RT 3.5 min
RT 4.5 min
2 Lys
0 Lys
1 Lys
0
20
40
60
80
100148767.8
148511.9148929.2
148639.7
148673.3148801.2
148835.7 149092.6148964.9
148564.0149031.6 149199.3148311.6 149364.3 149481.5
148300 148500 148700 148900 149100 149300 149500
Mass
0 2 4 6 8Time (min)
0
20
40
60
80
100
Rela
tive A
bundance Sliding window deconvolution
with RespectTM algorithm
Rela
tive I
nte
nsity
[G0F/G0F] 2 Lys
[G0F/G1F] 2 Lys
[(G0F/G2F)/(G1F)2] 2 Lys
-Lys
-Lys
Deconvoluted MS spectrum
Theoretical and experimental masses of infliximab
Glycoforms of lysine variants
Lys-Variant Glycoform Theor. average mass Exp. average mass ∆ Mass (ppm)
2 Lys G0F/G1F 148930.7 148929.2 9.8
2 Lys G0F/G0F 148768.5 148767.8 4.8
2 Lys (G1F)2 or (G0F/G2F) 149092.8 149092.6 1.3
1 Lys G0F/G0F 148640.3 148639.7 4.3
1 Lys G0F/G1F 148802.5 148801.2 8.6
1 Lys (G1F)2 or (G0F/G2F) 148964.6 148964.9 -1.8
0 Lys G0F/G0F 148512.2 148511.9 1.6
0 Lys G0F/G1F 148674.3 148673.3 6.8
0 Lys (G1F)2 or (G0F/G2F) 148836.5 148835.7 5.1
The ZipChip system coupled with the Q
Exactive MS BioPharma platform is
unique and powerful to separate and identify different intact antibody charge
variants in native and denatured
conditions
• Baseline separation of intact mAb
charge variants resulting from different
levels of Lys-clipping can be achieved
within three minutes by ZipChip system
• High resolution accurate mass spectra
of all lysine variants are confidently detected on Q Exactive Plus/HF/HF-X
MS with BioPharma option
• Three major glycoforms from each of the three lysine variants are identified
by BioPharma Finder with mass
accuracies better than 10 ppm
3 ng of sample injected
Intact mAb - Infliximab Analysis in HMR mode
MS data was acquired on a Q Exactive HF-X MS
with BioPharma OptionCE separation was achieved on ZipChip HR
with 10mM Ammonium Acetate in 10% Isopropanol as BGE
Resolution: 30K
16
The rapid separation and accurate
identification of highly differently
abundant charge variants can also be
consistently achieved by the ZipChipsystem and Q Exactive BioPharma
platform
• Near baseline separation of intact NIST mAb charge variants with abundance
ranging over 2 orders of magnitudes can
be achieved by the ZipChip system
• High resolution accurate mass data of
each Lysine variant is confidently
obtained on Q Exactive Plus/HF/HF-X MS
• Glycoform with abundance as low as
0.16% of the base peak can be detected and identified
• All 5 major glycoforms from each of the
three different Lysine variants are identified by BioPharma Finder
Intact NIST mAb Analysis in HMR Mode
4920 4940 4960 4980
m/z
0
20
40
60
80
100
0
20
40
60
80
100
Rela
tive A
bundance
0
20
40
60
80
1004941.03
4946.44
4935.68
4951.80
4957.22
4945.27
4950.62
4939.84
4956.07
4961.46
4949.53
4944.11
4960.26
4965.69
NIST mAb + 0 Lys
NIST mAb + 1 Lys
NIST mAb + 2 Lys
+30
2.0 3.0Time (min)
20
40
60
80
100
Rela
tive A
bunda
nce
2.55
2.52
2.49
0
0 Lys
1 Lys
2 Lys
4600 4800 5000 5200 5400
m/z
0
50
100
0
50
100
Rela
tive A
bundance
0
50
1004941.03
4781.69
5111.40
4632.30
4957.22
5293.89
4802.31
4945.27
5115.804785.75
5298.444636.194966.75 5138.04
4806.46
4949.535120.25
4789.87
5308.79
4640.21
4971.14
4810.97
5142.36
0.65nL of 0.1ug/ul
NIST mAb injected
Protein Name Modification Average Mass
(Da)
Theoretical Mass
(Da)
Matched Mass Error
(ppm)
Intensity Relative
Abundance
RT
(min)
NIST 1xG0F_G0F 148038.8 148037.1 11.5 1.28E+09 58.82
NIST 1xG0F_G1F 148200.0 148199.3 5.0 2.18E+09 100.00
NIST 1xG1F_G1F 148362.1 148361.2 6.0 1.82E+09 83.26
NIST 1xG1F_G2F 148522.7 148523.5 5.8 1.00E+09 46.00
NIST 1xG2F_G2F 148684.6 148685.7 7.2 4.84E+08 22.21
NIST_plus1K 1xG0F_G0F 148165.6 148165.3 1.9 7.66E+07 3.51
NIST_plus1K 1xG0F_G1F 148327.4 148327.4 0.1 1.39E+08 6.36
NIST_plus1K 1xG1F_G1F 148489.7 148489.4 2.5 1.18E+08 5.40
NIST_plus1K 1xG1F_G2F 148652.3 148651.7 3.6 5.97E+07 2.74
NIST_plus1K 1xG2F_G2F 148811.9 148813.9 13.4 2.90E+07 1.33
NIST_plus2K 1xG0F_G0F 148295.4 148293.5 13.1 1.04E+07 0.48
NIST_plus2K 1xG0F_G1F 148456.9 148455.6 9.0 1.56E+07 0.72
NIST_plus2K 1xG1F_G1F 148618.4 148617.5 5.6 1.51E+07 0.69
NIST_plus2K 1xG1F_G2F 148779.4 148779.9 3.1 8.07E+06 0.37
NIST_plus2K 1xG2F_G2F 148942.0 148942.0 0.4 3.49E+06 0.16
2.42
2.49
2.55
Similar MS data were obtained on both Q Exactive Plus MS and Q
Exactive HF MS equipped with BioPharma OptionCE separation was achieved on ZipChip HR
with Intact Antibody Assay Kit
65pg of sample
injected
Resolution: 17,500
17
0
50
100150931.7
151091.8152048.7
151888.7
151252.7149974.5 150134.3
153007.0152210.5
150294.9152845.5
153168.3149176.3149015.4
151314.3153964.5149336.8
154126.3150358.5 152272.1151411.8 152371.5 153802.9150453.7154921.9148220.6
150580.1 153328.4 155143.3151538.8149499.1 154150.3149559.6148379.0 154758.8 155308.0
148871.3
154185.4152497.4
154282.5
148440.6 151744.1 155805.4153467.9 154661.2
155718.9
155474.7
154922.8
148500 150000 151500 153000 154500 156000
2.0 3.0 4.0 5.00
50
1003.34
1.0
Heterogenetic ADCs can be
successfully characterized within 1
minute without sample pre-treatment
by the ZipChip system and Q Exactive BioPharma platform, and
the powerful BioPharma Finder
software
• Powerful sliding window capability
enabled by BioPharma Finder
software
• All of the different forms of
Trastuzumab Emtansine can be
analyzed in less than 40 seconds with only 3 nanograms of sample
injected
• No sample pre-treatment required
• The calculated average DAR value
is consistent with previous published
data
Antibody-Drug Conjugate (ADC) Analysis in HMR Mode
4600 4650 4700 4750 4800 4850 4900 4950 5000 5050 5100 5150 5200 5250 5300 5350 5400 5450 55000
50
1005037.42
4874.895005.444905.77
4844.05 5069.29
5042.76
4911.01
4967.86
5074.72 5178.01 5205.514722.68 5101.284752.56 4813.01 5106.594782.45
5244.085145.03
5249.654692.75 5133.15
5271.445277.07
4995.22
5362.97 5391.495282.564608.56 5397.234637.63 5328.825304.57
4657.80 5431.315436.84
5471.365499.94
Electropherogram of Trastuzumab Emtansine
Deconvoluted spectrum
m/z
Re
lative
Ab
un
dan
ce
DAR 0 DAR 1 DAR 2 DAR 3 DAR 4 DAR 5 DAR 6 DAR 7 DAR 8
G0F/G1F DAR Form Mass Accurancy (ppm) Relative abundance
DAR0 10.72 9.95
DAR1 7.45 33.47
DAR2 1.23 58.97
DAR3 2.37 79.90
DAR4 5.63 69.92
DAR5 9.08 49.14
DAR6 13.89 28.97
DAR7 9.93 12.64
DAR8 8.41 2.83
Average Drug-to-Antibody Ratio (DAR) is 3.47
MS data was acquired on a Q Exactive HF with BioPharma
Option
CE separation was achieved on ZipChip HR with Intact
Antibody Assay Kit
Resolution: 15KMS spectrum
18
Fast, sensitive, and accurate antibody
subunit analysis can be
accomplished by the ZipChip system
and Thermo Scientific Q ExactivePlus/HF/HF-X MS platform
• Separation of infliximab mAb
subunits can be achieved in 3 minutes by ZipChip system
• The sliding window method combined
with Thermo Scientific™ Xtract™ deconvolution algorithm in
BioPharma Finder software enables
monoisotopic mass determination of each subunit
• Lysine variants and their major
glycoforms of the subunits can be identified by BioPharma Finder
software
IdeS digested + reduced infliximabIdeS digested infliximab
Subunit Monoisotopic theo. mass Experimental mass ∆ Mass (ppm)
Light chain 23424.3946 23424.4671 3.1
Fd' 25631.5325 25631.5644 1.2
Fc/2 G0F 25316.5863 25316.6345 1.9
Fc/2 G1F 25478.6391 25478.5753 2.5
(Fc/2 G0F) -Lys 25188.49131 25188.5763 3.3
(Fc/2 G1F) -Lys 25350.54413 25350.5246 0.8
F(ab')2 98146.3 (av.) 98146.8 -4.8
Fc/2 G0F (2 SS bonds) 25312.5550 25312.5524 0.1
Fc/2 G1F (2 SS bonds) 25474.6078 25474.5138 3.7
(Fc/2 G0F) -Lys (2 SS bonds) 25184.4600 25184.4311 1.1
(Fc/2 G1F)- Lys (2 SS bonds) 25346.5128 25346.4730 1.6
2.0 2.2 Time (min)0
20
40
60
80
100
Rela
tive A
bundance
2.11
2.14
2.26
(Fc/2) -Lys
F(ab’)2
Fc/2
120 k15 k
z=44
z=45 z=42
z=43
2000 2500m/z
0
20
40
60
80
100
Rela
tive A
bundance
2283.5
2231.7
2182.0z=41
z=46
z=48
Deconvolutionwith Respect 98146.8
98000 100000Mass
0
25312.5524
25184.4311
25474.513825346.4730
25100 25300 25500Mass
20
40
60
80
100
Rela
tive I
nte
nsity
(Fc/2 G0F) -Lys
Fc/2 G0F
(Fc/2 G1F) -LysFc/2 G1F
F(ab’)2
2.5 3.0 3.5 Time (min)
0
20
40
60
80
100
Rela
tive A
bundance 3.42 3.48
3.06
LC
Fd’(Fc/2) -LysFc/2
120 k
800 900 1000 1100m/z
0
50
100
0
50
100
Rela
tive A
bundance
845.43905.71
975.301014.31
1102.43
901.17970.37
1009.23
1096.90
841.13 (Fc/2) -Lys
Fc/2
LC
Fd’
1000 1500m/z
0
50
100
0
50
100
Rela
tive A
bundance
885.41 1026.87
1069.61
1166.81
938.54
1066.35
1172.93
1303.14
869.10
23424.467125631.5644
20
40
60
80
100
Rela
tive I
nte
nsity
Mass 2500024000
Fd’LC
(Fc/2 G0F) -Lys
Fc/2 G0F
(Fc/2 G1F) -LysFc/2 G1F
0
1200 1400m/z
0
50
100
0
50
100
Rela
tive A
bundance
1327.21401.1
1261.0
1483.31201.0
(Fc/2) -Lys
1334.11267.4
1408.21207.0
1490.9
Fc/2
IdeS
dig
est
IdeS
dig
est
plu
s r
eduction
mAb Subunits Analysis in Protein mode
MS data was acquired on a Q Exactive HF-X MS
with BioPharma OptionCE separation was achieved on ZipChip HR
with Peptides Assay Kit
19
0
50
100
1 2 3 4 5 6 7 8 9 10 11
The combination of ZipChip sample
separation, Q Exactive Plus/HF/HF-X
MS platform produced HRAM MS and
MS/MS spectra, and BioPharma Finder software enables fast and accurate
peptide identification
• Plug and play ZipChip delivers stable nano spray and nano spray level
sensitivity
• CE-MS/MS analysis can be completed in 10 minutes
• Only a few nanograms of sample are sufficient for the analysis
• 98% sequence coverage based on
MS/MS data for the light chain and heavy chain is confidently achieved
ProteinsNumber of MS
Peaks
MS Peak
Area
Sequence
CoverageAbundance
NSIT mAb light chain 141 26.4% 100.0% 41.67%
NIST mAb heavy chain 339 60.5% 97.6% 56.35%
Unidentified 1441 12.6%
1.2 ng of sample injected
NIST mAb light chain
NIST mAb heavy chain
Unidentified
Peptide Mapping Analysis in Standard Mode
Re
lative
Ab
un
dan
ce
Time (Min)
Electropherogram of Digested NIST mAb
MS data was acquired on a Q Exactive Plus MS with
BioPharma Option
CE separation was achieved on ZipChip HR with Peptides
Assay kit
MS resolution: 60K
MS/MS resolution: 15k
20
Content
Resources and Ordering Information
ZipChip System Introduction
ZipChip Separation Mechanism
Biopharmaceutical Application Examples
Metabolomics Application and System Test
21
Complete 20 Amino Acids Analysis
Extremely rapid separation and
accurate identification of all 20 amino
acid mix can be confidently achieved by
the ZipChip system and Thermo Scientific Q Exactive
/Focus/Plus/HF/HF-X MS platform
• Separation of 20 amino acids can be completed in 2 minutes by the ZipChip
system
• Isomers, Leucine(L) and Isoleucine(I), can be baseline separated
• Co-elueted amino acids can be identified with HRAM mass
spectrometry
• Femto grams level of sample is sufficient for each analysis
MS data was acquired on a Q Exactive MS
CE separation was achieved on ZipChip HS with Metabolites
Assay kit
Electropherogram of 10uM Promega 20 AA Mix
1.0 1.2 1.4 1.6 1.8 2.0 2.2Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bu
nd
an
ce
0.95
0.991.63
1.351.60
1.68
1.82
1.201.87
Base Peak
20 AA Extraction
1.54
1.72
K
R
H
GA
V IL
S
M,T
0.90
N,P
F
W,QY
E,CD
120 125 130 135 140 145 150 155 160 165 170 175 180m/z0
50
100
Rela
tiv
e A
bu
nd
an
ce
150.05851
120.06568
M
T
140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230m/z
0
50
100205.09689
147.07660wQ
116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134m/z0
50
100116.07079
133.06086
N
P
122 124 126 128 130 132 134 136 138 140 142 144 146 148m/z0
50
100 148.06065
122.02702C
E
1.381.41
I L
22
Linear Dynamic Range Test of Arginine and Valine
y = 1E+08x + 2E+07R² = 0.998
0,E+00
2,E+08
4,E+08
6,E+08
8,E+08
1,E+09
1,E+09
0 2 4 6 8 10 12P
ea
k A
reaConcentration (μM)
Arginine (R) 10nM~10μM
y = 1E+08x + 4E+06R² = 0.9999
0,E+00
2,E+08
4,E+08
6,E+08
8,E+08
1,E+09
1,E+09
0 2 4 6 8 10 12
Pe
ak
Are
a
Concentration (μM)
Valine (V) 10nM~10μM
Minimum sample loading of 40
attomole Arginine and Valine can be
achieved by the ZipChip system and
Thermo Scientific Q Exactive/Focus/Plus/HF/HF-X MS platform
• Arginine and Valine calibration curves
provide good linearity over the range of 10 nM to 10 µM
• Lowest sample injection amount on
the linear curve is 40 attomoles (4nL of 10 nM sample injected)
MS data was acquired on a Q Exactive MS
CE separation was achieved on ZipChip HS with
Metabolites Assay kit
23
Quick Analysis of Amino Acids in Gibco™ DMEM Cell Culture Media
K
R
H
G
V
I
L
S
M, TF
0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2
Time (min)
0
20
40
60
80
100
Re
lative
Ab
un
dan
ce
0.83
147.11197
1.70147.075581.35
132.10205
0.64
157.08342 1.38132.102050.88
175.118701.32
118.08601
1.64
166.086001.55
120.066160.69
105.003790.92
156.07758
1.84
182.081851.44
106.05044 1.94
241.028920.75
104.107061.07
76.03958
NL: 4.49E8
Base Peak MS Q
YW1.67
205.09830
choline
The ZipChip system and Thermo
Scientific Q Exactive
/Focus/Plus/HF/HF-X MS platform can
rapidly separate and identify the amino acids presented in cell culture
media
• Sample can be directly analyzed after simple filtration and dilution
• On chip desalting capability allows
direct analysis of samples in high salt condition without pre-treatment
• Separation can be completed in 2 minutes by the ZipChip system
• All 15 amino acids presented in the
cell culture media are identified and results match up with the aa
composition indicated in the
commercial sample spec sheet
MS data was acquired on a Q Exactive MS
CE separation was achieved on ZipChip HS with
Metabolites Assay kit
C-C
24
Calibration Curve of Internal Standard in Cell Culture Media
y = 2E+07x + 484283R² = 0.9996
0,E+00
5,E+07
1,E+08
2,E+08
2,E+08
3,E+08
0 2 4 6 8 10 12
Sig
na
l In
ten
sit
y
Concentration (μM)
Concentration range: 50 nM to 10μM
Val-d8Accurate quantification of amino
acids in cell culture media with stable
isotope labeled internal standard can
be achieved by the ZipChip system and Thermo Scientific Q Exactive
/Focus/Plus/HF/HF-X platform
• Deuterated Valine standard, Valine-d8, was spiked into the crude cell
culture media
• High linearity calibration curve with concentration range from 50nM to
10µM can be established.
MS data was acquired on a Q Exactive Classic MS
CE separation was achieved on ZipChip HS with Metabolites
Assay kit
25
Carryover Test by Monitoring Arginine
0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Rela
tive A
bundance
0
20
40
60
80
100
0.95175.119
0.99156.077 1.34
118.0861.60
116.0711.37
132.1021.53
150.058 1.64205.097 1.78
182.0811.2090.055
1.84134.045
1.43106.050
0.95175.119
0.96175.119
NL: 1.51E9
Base Peak MS
NL: 1.51E9Base Peak m/z=175.118-175.120 MS
NL: 1.43E8
Base Peak MS
NL: 8.21E5Base Peak m/z=175.118-175.120 MS
10µM AA mix
Extraction of Arg
Extraction of Arg
Blank
The ZipChip system has extremely
low carryover
• Blank injections interleaved between 10 µM AA mix injections
• Extracted ion electropherogram of
Arginine from sample injection and blank injection were used to calculate
the carryover
• Observed carryover of Arginine is only 0.054% or less
MS data was acquired on a Q Exactive Classic MS
CE separation was achieved on ZipChip HS with Metabolites
Assay kit
26
Migration Time Reproducibility Test Using Arginine (R) Standard
0,5
0,7
0,9
1,1
1,3
1,5
1 11 21 31 41 51 61 71 81 91
Mig
rati
on
Tim
e (
min
)
Injections
1st to 50th injections from a single vial RSD= 1.76%
51st to 100th injections from 96-well plate RSD= 0.84%
RSD= 1.85% (1st to 100th injections)
RSD=0.54% (8th to 50th injections)
Operation of the ZipChip system
through ZipChip autosampler can
achieve good reproducibility
• The migration time of Arginine was
monitored throughout 100 runs; the
overall RSD value of the migration
time is 1.85%
• It takes 5-10 injections for the system
to get stabilized
• First 50 injections are from the same
sample vial; the RSD value of the
migration time is 1.76%; the RSD value dropped to 0.54% if excludes
the first 7 injections
• Last 50 injections are from 50 different sample wells; the RSD value
of the migration time is only 0.84%
MS data was acquired on a Q Exactive MS
CE separation was achieved on ZipChip HS with Metabolites
Assay kit
27
Content
Resources and Ordering Information
ZipChip System Introduction
ZipChip Separation Mechanism
Biopharmaceutical Application Examples
Metabolomics Application and System Test
28
Consumable Spec sheet
Application Note
www.thermofisher.com/zipchip
Hardware Spec Sheets
CE/ESI-MS Brochure
Available Resources
908devices.com/products/zipchip
29
Ordering Information
Product P/N
Hardware
ZipChip Interface (Autosampler version) 00950-01-00492
ZipChip Interface (Manual version) 00950-01-00493
ZipChip Autosampler 00950-01-00494
DryDock 00950-01-00518
Consumables
Installation Test Kit 00950-01-00510
ZipChip HS 00950-01-00498
ZipChip HR 00950-01-00499
ZipChip Metabolites Assay Kit 00950-01-00500
ZipChip Peptides Assay Kit 00950-01-00501
ZipChip Intact Antibody Assay Kit 00950-01-00502
30
Thank you!
Any Questions?
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