particularly improved the ability for identification of lower abundance phosphopeptides [27].
In this chapter, we describe a detailed “ShortPhos” workflow
for phosphopeptides enrichment from plant membrane proteins.
We highlight critical steps in the preparation of plant membranes
for phosphoproteome analysis. In this protocol, there are three
major improvements: First, we optimized membrane protein
extraction by using Dounce Homogenizer-based membrane-fraction (MF) isolation instead of using the traditional mortar and
pestle, resulting in higher yields of membrane proteins. Secondly,
we optimized the processing of phosphopeptide enrichment.
Finally, we omitted the fractionation of peptides and performed a
single-MS run per sample, resulting in less hands-on time as compared with traditional methods. This current protocol significantly
improved the ability to identify low abundance phosphopeptides.
2 Materials
2.1 Buffers and
Solutions
1. Homogenization buffer (HB): 330 mM Sucrose, 100 mM
KCl, 1 mM EDTA, 50 mM Tris-MES, pH 7.5. Dissolve
113 g sucrose, 7.5 g KCl, 0.37 g EDTA, and 6.06 g Tris base
into 900 mL deionized water, mix well, and adjust pH to 7.5
with 2 M MES (see Note 1). Fill water until 1 L. Store at 4
C
for short-time (maximal 5 days) use or at À20
C for long-term
storage. Add 5 mM DTT (see Note 1), protease inhibitor
cocktail (50 μL/10 mL HB), phosphatase inhibitor cocktail
2 (50 μL/10 mL HB), and phosphatase inhibitor cocktail
3 (50 μL/10 mL HB) immediately before use.
2. Tris–HCl: 10 mM, pH 8.0. Dissolve 1.21 g Tris base into
900 mL deionized water, mix well, and adjust pH till 8.0
with 1 N HCl. Fill with water until 1 L.
3. UTU: 6 M Urea and 2 M Thiourea, pH 8.0. Dissolve 3.6 g
urea, 1.5 g thiourea into 9 mL 10 mM Tris–HCl solution
(pH 8.0), and mix well. Fill Tris–HCl solution (pH 8.0) until
10 mL, mix well. This solution should be freshly prepared.
4. DTT Reduction buffer: 1 μg/μL DTT in water. Dissolve
10 mg DTT into 10 mL of deionized water (see Note 2).
5. Alkylation buffer (IAA): 5 μg/μL iodoacetamide (IAA) in
water. Dissolve 50 mg iodoacetamide in 10 mL deionized
water (see Note 2).
6. Resuspension buffer for phosphopeptide enrichment: 1 M glycolic acid in 80% acetonitrile (ACN) and 6% trifluoroacetic acid
(TFA). Mix 8 mL ACN, 0.6 mL TFA, and 1.4 ml deionized
water well, and then dissolve 0.76 g glycolic acid into this
mixture (see Note 3).
Plant Membranes Phosphoproteome
443
In this chapter, we describe a detailed “ShortPhos” workflow
for phosphopeptides enrichment from plant membrane proteins.
We highlight critical steps in the preparation of plant membranes
for phosphoproteome analysis. In this protocol, there are three
major improvements: First, we optimized membrane protein
extraction by using Dounce Homogenizer-based membrane-fraction (MF) isolation instead of using the traditional mortar and
pestle, resulting in higher yields of membrane proteins. Secondly,
we optimized the processing of phosphopeptide enrichment.
Finally, we omitted the fractionation of peptides and performed a
single-MS run per sample, resulting in less hands-on time as compared with traditional methods. This current protocol significantly
improved the ability to identify low abundance phosphopeptides.
2 Materials
2.1 Buffers and
Solutions
1. Homogenization buffer (HB): 330 mM Sucrose, 100 mM
KCl, 1 mM EDTA, 50 mM Tris-MES, pH 7.5. Dissolve
113 g sucrose, 7.5 g KCl, 0.37 g EDTA, and 6.06 g Tris base
into 900 mL deionized water, mix well, and adjust pH to 7.5
with 2 M MES (see Note 1). Fill water until 1 L. Store at 4
C
for short-time (maximal 5 days) use or at À20
C for long-term
storage. Add 5 mM DTT (see Note 1), protease inhibitor
cocktail (50 μL/10 mL HB), phosphatase inhibitor cocktail
2 (50 μL/10 mL HB), and phosphatase inhibitor cocktail
3 (50 μL/10 mL HB) immediately before use.
2. Tris–HCl: 10 mM, pH 8.0. Dissolve 1.21 g Tris base into
900 mL deionized water, mix well, and adjust pH till 8.0
with 1 N HCl. Fill with water until 1 L.
3. UTU: 6 M Urea and 2 M Thiourea, pH 8.0. Dissolve 3.6 g
urea, 1.5 g thiourea into 9 mL 10 mM Tris–HCl solution
(pH 8.0), and mix well. Fill Tris–HCl solution (pH 8.0) until
10 mL, mix well. This solution should be freshly prepared.
4. DTT Reduction buffer: 1 μg/μL DTT in water. Dissolve
10 mg DTT into 10 mL of deionized water (see Note 2).
5. Alkylation buffer (IAA): 5 μg/μL iodoacetamide (IAA) in
water. Dissolve 50 mg iodoacetamide in 10 mL deionized
water (see Note 2).
6. Resuspension buffer for phosphopeptide enrichment: 1 M glycolic acid in 80% acetonitrile (ACN) and 6% trifluoroacetic acid
(TFA). Mix 8 mL ACN, 0.6 mL TFA, and 1.4 ml deionized
water well, and then dissolve 0.76 g glycolic acid into this
mixture (see Note 3).
Plant Membranes Phosphoproteome
443