over Amicon 30 kDa MWCO filter, and 1 min at 4
C at
16,000 Â g to remove potential aggregates) to avoid clogging
of the column.
10. Before performing the actual separation of the target sample it
is important to define the void volume of the system (void of
HPLC and void of column) and to define at which percentage
of column volume sample has to be collected (start of fractionation). We propose to estimate the value using low concentrated Blue Dextran solutions (if the column separates until
1.5 MDa, Yarra SEC4000).
11. Fractions from SEC-separations can be stored in the fridge
overnight and the sample workup steps can continue the following day. Alternatively, collected fractions can be stored for
several weeks at À80
C. For freezing the samples, fractionate
them in a 96 well plate (e.g., Agilent, 203426-100), as the
FASP-filter plate should not be frozen.
12. Depending on the HPLC instrument setup, it is possible that
the filter plate could not be compatible with the fraction collector. Therefore, fractionation can be done on suitable 96 well
plate and samples can be transferred in a second step in the
96-Well Filter Plates for MS sample processing.
13. It is important to accurately wash the filter plate with twice
200 μL of HPLC-grade H 2 O until there is no volume left
(>5 μL per plate); the manufacturer passivated the filter-plates
with glycerin and not appropriate washing step can result in
severe contamination of MS samples and compromise the spray
quality during MS analysis.
14. Plate filtration is not always equal across samples, and it is
necessary to check that after every centrifugation step that the
volume is removed. To have a more homogenous filtration
step, it is recommended to split every centrifugation step into
2 parts where the filter plate is in 2 different positions. Before
the second step, the filter plate is reflected along the long side
(i.e., A12 will face outside in the centrifuge once and then
inside).
15. The volume after the last washing step with buffer 100 μL ABC
(1800 Â g for 60 min) must be less than 10 μL. If there is more
leftover volume, increase the centrifugation time. It is important to have less than 10 μL sample volume to allow more
homogenous proteolysis of all samples in the wells and to
avoid the presence in the proteolysis buffer of urea, TCEP,
and iodoacetamide that can compromise the spray quality during the MS analysis.
16. The loading can be heterogeneous for some of the fractions. If
there is some sample left after the centrifugation, repeat the
centrifugation until the sample loading is complete.
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Andrea Fossati et al.
C at
16,000 Â g to remove potential aggregates) to avoid clogging
of the column.
10. Before performing the actual separation of the target sample it
is important to define the void volume of the system (void of
HPLC and void of column) and to define at which percentage
of column volume sample has to be collected (start of fractionation). We propose to estimate the value using low concentrated Blue Dextran solutions (if the column separates until
1.5 MDa, Yarra SEC4000).
11. Fractions from SEC-separations can be stored in the fridge
overnight and the sample workup steps can continue the following day. Alternatively, collected fractions can be stored for
several weeks at À80
C. For freezing the samples, fractionate
them in a 96 well plate (e.g., Agilent, 203426-100), as the
FASP-filter plate should not be frozen.
12. Depending on the HPLC instrument setup, it is possible that
the filter plate could not be compatible with the fraction collector. Therefore, fractionation can be done on suitable 96 well
plate and samples can be transferred in a second step in the
96-Well Filter Plates for MS sample processing.
13. It is important to accurately wash the filter plate with twice
200 μL of HPLC-grade H 2 O until there is no volume left
(>5 μL per plate); the manufacturer passivated the filter-plates
with glycerin and not appropriate washing step can result in
severe contamination of MS samples and compromise the spray
quality during MS analysis.
14. Plate filtration is not always equal across samples, and it is
necessary to check that after every centrifugation step that the
volume is removed. To have a more homogenous filtration
step, it is recommended to split every centrifugation step into
2 parts where the filter plate is in 2 different positions. Before
the second step, the filter plate is reflected along the long side
(i.e., A12 will face outside in the centrifuge once and then
inside).
15. The volume after the last washing step with buffer 100 μL ABC
(1800 Â g for 60 min) must be less than 10 μL. If there is more
leftover volume, increase the centrifugation time. It is important to have less than 10 μL sample volume to allow more
homogenous proteolysis of all samples in the wells and to
avoid the presence in the proteolysis buffer of urea, TCEP,
and iodoacetamide that can compromise the spray quality during the MS analysis.
16. The loading can be heterogeneous for some of the fractions. If
there is some sample left after the centrifugation, repeat the
centrifugation until the sample loading is complete.
290
Andrea Fossati et al.