reference sample over the fragmentation cycle was established as a
method called ultra definition mass spectrometry (UDMS
E ).
UDMS
E provides better coverage of complex proteomes and allows
for better control of collision energy, while still keeping the fragmentation benefits of a traveling-wave ion mobility mass spectrometry [25–27]. UDMS
E has been shown to improve the efficiency of
fragmentation, increasing peptide identification rates, and consequently leading to greater sample coverage [26]. Also important, as
in several other methods, UDMS
E is compatible with relative
quantitation, since the relationship between MS signal response
and protein concentration can be used to compare protein abundance across analyzed samples, a fact that allows the identification
of potential protein biomarkers [28].
UDMS
E can contribute to improve the body knowledge of
multifactorial diseases and propose potential markers by obtaining
a reliable identification of a high number of proteins across different
groups of human blood plasma. There are still few studies in both
individual and pooled samples employing UDMS
E to investigate
biological and molecular mechanisms of complex diseases or potential biomarkers of diagnosis or response to treatment. Thus, we
present a step-by-step protocol to assist in the identification of
proteins in human blood plasma, to contribute to possible further
studies using such technology.
2 Materials
2.1 Liquid
Chromatography
Equipment
and Software
1. HPLC system (Waters Corporation).
2. Absorbance detector (Waters Corporation).
3. Injection system.
4. Buffer solution A: 40 mM sodium phosphate (pH 7.4), 0.5 M
NaCl, 0.02% sodium azide.
5. Buffer solution B: 2 M Urea, 0.5 M glycine (pH 2.25).
6. Empower Pro 2 software (Waters Corporation).
7. MARS Hu14 column (Agilent Technologies) (4.6 mm inner
diameter, length 100 mm, capacity of 40 μL of plasma or
serum).
2.2 Mass
Spectrometry
Equipment
and Software
1. Waters Total Recovery vials with preslit PTFE/silicone caps.
2. Acquity UPLC M-Class with 2D RPxRP technology (Waters
Corporation).
3. Synapt G2-Si Mass Spectrometer (Waters Corporation).
4. Acquity UPLC M-Class Peptide BEH C18 Trap Column
(130 A ˚ , 5 μm, 300 μm  50 mm) (Waters Corporation).
156
Licia C. Silva-Costa et al.
method called ultra definition mass spectrometry (UDMS
E ).
UDMS
E provides better coverage of complex proteomes and allows
for better control of collision energy, while still keeping the fragmentation benefits of a traveling-wave ion mobility mass spectrometry [25–27]. UDMS
E has been shown to improve the efficiency of
fragmentation, increasing peptide identification rates, and consequently leading to greater sample coverage [26]. Also important, as
in several other methods, UDMS
E is compatible with relative
quantitation, since the relationship between MS signal response
and protein concentration can be used to compare protein abundance across analyzed samples, a fact that allows the identification
of potential protein biomarkers [28].
UDMS
E can contribute to improve the body knowledge of
multifactorial diseases and propose potential markers by obtaining
a reliable identification of a high number of proteins across different
groups of human blood plasma. There are still few studies in both
individual and pooled samples employing UDMS
E to investigate
biological and molecular mechanisms of complex diseases or potential biomarkers of diagnosis or response to treatment. Thus, we
present a step-by-step protocol to assist in the identification of
proteins in human blood plasma, to contribute to possible further
studies using such technology.
2 Materials
2.1 Liquid
Chromatography
Equipment
and Software
1. HPLC system (Waters Corporation).
2. Absorbance detector (Waters Corporation).
3. Injection system.
4. Buffer solution A: 40 mM sodium phosphate (pH 7.4), 0.5 M
NaCl, 0.02% sodium azide.
5. Buffer solution B: 2 M Urea, 0.5 M glycine (pH 2.25).
6. Empower Pro 2 software (Waters Corporation).
7. MARS Hu14 column (Agilent Technologies) (4.6 mm inner
diameter, length 100 mm, capacity of 40 μL of plasma or
serum).
2.2 Mass
Spectrometry
Equipment
and Software
1. Waters Total Recovery vials with preslit PTFE/silicone caps.
2. Acquity UPLC M-Class with 2D RPxRP technology (Waters
Corporation).
3. Synapt G2-Si Mass Spectrometer (Waters Corporation).
4. Acquity UPLC M-Class Peptide BEH C18 Trap Column
(130 A ˚ , 5 μm, 300 μm  50 mm) (Waters Corporation).
156
Licia C. Silva-Costa et al.