Quantification of Proteins by Label-Free LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 219

quantification of hundreds of proteins with a high dynamic range
across more than three orders of magnitude ( 3 , 15 ) . Further-
more, the reproducibility of the platform is high enough to per-
form accurate relative quantitation of proteins across dozens of
samples at a time, which makes this approach ideal for large-scale
biomarker discovery as well as systems biology ( 4 ) .
The protocol for label-free LC-MS/MS includes three steps:
( 1 ) nanoLC- MS E conditions, ( 2 ) data processing/database
search, and ( 3 ) data analysis. The first part includes informa-
tion of optimum nano liquid chromatography and mass spec-
trometry conditions for analysis of complex samples. The second
part describes the data processing and database searching param-
eters. The last part describes the steps needed to analyze a typical
data set.

2. Materials

1. Liquid Chromatography: 10kpsi nanoAcquity; trapping col-
umn: Symmetry C18 180

μ m × 20 mm 5 μ m particles;

Analytical column: BEH 75 μ m × 200 mm 1.7 μ m parti-

cles (Waters Corp., Milford, MA).
2. Mass Spectrometer: Q-Tof Premier (Waters Corp., Milford,
MA).
3. For all preparations and mobile phase HPLC grade water
and acetonitrile were used (water - Sigma; acetonitrile -
Fisher).
4. Digested yeast enolase (Waters, Milford, MA).
5. Four protein digest mix 1 & 2 (Waters, Milford, MA).
6. Processing, database searching, and time alignment software
package.

3. Methods

3.1.Liquid
Chromatography-
Mass
Spectrometry

The protocol for label-free quantitation of proteins by LC-MS is
a 'bottom-up' approach, which means proteins must be digested
into peptides. The analytical process is then performed at the pep-
tide level.
Each biological sample should be injected and analyzed in
triplicate followed by a blank injection (to ensure there is no car-
ryover of peptides from one sample to the other in this sequential