nano LC, proteomics, LC MS, 2D LC EASY-nLC

Excellent high resolution results – every time

Accurate label-free quantification ensured by minimal RSD values for retention times
Stable, pulsation-free gradients from 100 nL/min
Advanced Flow Control™ with two flow sensors prior to mixing
Split-free gradient mixing – no flow splitting

Fast separations with typical peak widths: 3 - 5 sec (FWHM)

With ThermoFisher LCQ mass spectrometer

System:

Sample:

Analytical column:

Gradient:

EASY-nLC

fmol tryptic BSA digest

EASY-Column, 10 cm, ID75 μm,
3 μm, C18-A2

300 nL/min, 5-35% B in 10 min
Solvent A: 0.1% formic acid
Solvent B: acetonitrile, 0.1% formic acid
FWHM ~3.5 sec

Low retention time RSD values for complex mixtures

With Thermo Fisher LTQ Orbitrap XL

FT scan 390-1500 m/z

Column:

Sample:

15 cm, 75 µm ID, C₁₈ 5 µm

100 fmol annexin V
800 fmol gelsolin
300 fmol BSA digestion mixture

Gradient:

300 nl/min, 3 - 35 %B in 25 minutes

Courtesy of J.M. Asara, Beth Israel Deaconess Medical Center, Boston, MA.

Excellent sensitivity

Excellent mass accuracy

With Thermo Fisher TSQ- Quantum Ultra
MRM scan (m/z 651.3) 200 attomole BSA
Injected sample: 2µl of 100 amol/µl BSA tryptic digest.
RT RSD 0.08%

Courtesy of Mr. Paul Taylor, Hospital for Sick Children, Ontario.

Superior sample handling

Efficient Peltier-controlled sample cooling
Injection volume selectable in 0.01 μl steps
Injection needle washed outside and inside
Custom wash with 2 extra solvent bottles

Low carryover

With Thermo Fisher LTQ XL with ETD

Column: 180 µm x 10 cm C18 column

Flow rate: 1 µL/min

Sample: Alternate injections (2 µL): angiotensin II, 1.0 pmol/µL or 0.1% formic acid (blank)

Loop and needle wash (standard procedure): 100 µL flush volume

Solvents: A = 0.1% formic acid; B = 0.1% formic acid in 99.9% acetonitrile

Data processing: Sum of areas of angiotensin II peaks [524.0 m/z (+2); 349.8 m/z (+3)] measured for sample and blank runs.
Carryover calculated as % angiotensin extracted ion peak area in blank compared to extracted ion peak area in previous angiotensin injection.

Data courtesy of Thermo Fisher Scientific, San Jose, USA

Sample pick-up linearity

Table Extracted ion currents for 474.79 Th ion

Experiment 1:
Column: 180 µm x 10 cm C18 column
Flow rate: 1 µL/min
Gradient: 0-60% B in 30 min

Experiment 2:
Column: 200 µm x 5 cm, Halo 2.7 µm particles, C18 column
Flow rate: 1.5 µL/min
Gradient: 0-60% B in 10 min

With Thermo Fisher LTQ XL with ETD

Sample:
0.1, 0.5, 1, 2.5, 5, 20, 50, 100 and 250 fmol
tryptic -digest horse myoglobin injected in triplicate

Solvents:
A = 0.1% formic acid; B = 0.1% formic acid in 100% acetonitrile

Data processing:
Areas of two peptides from myoglobin digest [ALELFR (sum of 748.4 (+1) and 374.7 (+2) and LFTGHPETLEK (sum of 636.3 (+2) and 424.6 (+3)] measured for each injection.
Evaluation used linear regression analysis for all data at and above limit of detection (LOD). Pearson coefficient calculated.

Data courtesy of Thermo Fisher Scientific, San Jose, USA

Sample pick-up precision

Samples:
0.1% formic acid placed in a 96-well PCR plate and in plastic vials with built-in inserts

Injections:
5 µL injections performed for 6 µL and 5 µL samples

Post-injection:
Remaining solution volume measured with a micropipette. Volume removed during injection calculated.

Data courtesy of Thermo Fisher Scientific, San Jose, USA

2D-enabled operation

Identify more proteins

2D experiments identified 3-4 times as many proteins as 1D control

Venn diagram of identified number of proteins from analyses using the EASY-nLC system 2D configuration, conventional 2D (quaternary split-flow solvent delivery) or a 1D configuration.

Refer to technical note 'Identify more proteins compared to conventional approaches' for more details