not only proteome changes but specific PTMs as well, including
glycosylations, phosphorylations and acetylations, and profiles of
protein–protein interactions (PPIs); see Anjo et al. [1]. Moreover,
SWATH-MS technology can be applied to a wide variety of human
biological samples, including different biofluids (such as urine,
serum, plasma, and tears), tissues (e.g., heart, liver, and kidney),
different types of cell lines, and cellular secretomes [1]. Furthermore, SWATH-MS has been applied to other species including
animal models extensively studied (e.g., mouse/rat and zebrafish).
Finally, it is interesting to indicate that SWATH-MS presents advantages to other label-free methods in clinical applications and long
cohorts of individuals: (1) it is a low-cost method; (2) it has a simple
sample preparation method; and (3) it can be applied to large
cohorts of samples [26].
This chapter presents the workflow used by our group to
prepare and analyze biological samples with a TripleTOF
® 6600+
system using an Eksigent
® 425 nano-LC and ekspert™ 400 autosampler, for the SWATH-MS experiment.
2 Materials
All solutions must be prepared using ultrapure water (Milli-Q
water) and analytical grade for reagents or HPLC or LC-MS quality
for solvents (see Note 1).
2.1 Methods for
Protein Extraction
2.1.1 Protein Extractions
from Different Body Fluids
Saliva Samples
1. Microfuge.
2. Eppendorf clear.
3. Protease inhibitor mixture (see Note 2).
4. RC DC™ Protein Assay.
5. Methanol (MeOH).
6. Chloroform (CHCl 3 ).
7. Ultrapure water.
Tear Samples
1. Microcapillary tubes, or Schirmer strip to tears collected.
2. Microfuge.
3. Eppendorf clear.
4. Vortex mixer.
5. Acetone.
6. 100 mM ammonium bicarbonate (Ambic) in Milli-Q water.
7. RC DC™ Protein Assay.
110
Maria del Pilar Chantada-Va ´ zquez et al.
glycosylations, phosphorylations and acetylations, and profiles of
protein–protein interactions (PPIs); see Anjo et al. [1]. Moreover,
SWATH-MS technology can be applied to a wide variety of human
biological samples, including different biofluids (such as urine,
serum, plasma, and tears), tissues (e.g., heart, liver, and kidney),
different types of cell lines, and cellular secretomes [1]. Furthermore, SWATH-MS has been applied to other species including
animal models extensively studied (e.g., mouse/rat and zebrafish).
Finally, it is interesting to indicate that SWATH-MS presents advantages to other label-free methods in clinical applications and long
cohorts of individuals: (1) it is a low-cost method; (2) it has a simple
sample preparation method; and (3) it can be applied to large
cohorts of samples [26].
This chapter presents the workflow used by our group to
prepare and analyze biological samples with a TripleTOF
® 6600+
system using an Eksigent
® 425 nano-LC and ekspert™ 400 autosampler, for the SWATH-MS experiment.
2 Materials
All solutions must be prepared using ultrapure water (Milli-Q
water) and analytical grade for reagents or HPLC or LC-MS quality
for solvents (see Note 1).
2.1 Methods for
Protein Extraction
2.1.1 Protein Extractions
from Different Body Fluids
Saliva Samples
1. Microfuge.
2. Eppendorf clear.
3. Protease inhibitor mixture (see Note 2).
4. RC DC™ Protein Assay.
5. Methanol (MeOH).
6. Chloroform (CHCl 3 ).
7. Ultrapure water.
Tear Samples
1. Microcapillary tubes, or Schirmer strip to tears collected.
2. Microfuge.
3. Eppendorf clear.
4. Vortex mixer.
5. Acetone.
6. 100 mM ammonium bicarbonate (Ambic) in Milli-Q water.
7. RC DC™ Protein Assay.
110
Maria del Pilar Chantada-Va ´ zquez et al.