Plasma sample preparation was performed
according the protocol
in the Supporting Methods, which was adapted
from 3 previous studies.17 (link)−19 (link) All steps in this protocol were
completed without the aid of any robotics. Briefly, 1 μL of
plasma (60–70 μg protein) was added to 24 μL of
SDC buffer (1% sodium deoxycholate, 10 mM TCEP, 40 mM chloroacetamide,
and 100 mM Tris-HCl pH 8.5) in either a 500 μL 96-well
Protein Lo-bind plate, or Protein Lo-bind 1.5 mL tubes (Eppendorf).
After sealing the tube or plate (with a silicone mat, Eppendorf),
samples were heated to 95 °C for 10 min at 1,000 rpm in an Eppendorf
Thermomixer-C with a ThermoTop (heated lid) to denature, reduce, and
alkylate proteins. Once cooled to RT and diluted 10-fold with water,
LysC and Trypsin were added (from 1 mg/mL stock solutions in either
water, or 50 mM acetic acid, respectively) to digest proteins at 1:100
ratio (each protease:protein, μg/μg) and digested at 37
°C for 16 h at 1000 rpm in an Eppendorf Thermomixer-C with a
ThermoTop (heated lid). An equal volume (250 μL) of 99% ethyl
acetate/1% TFA was added to the digested peptides for a final concentration
of 49.5% ethyl acetate and 0.5% TFA.
SDB-RPS StageTips were
generated by punching double-stacked SDB-RPS discs (Sigma, Cat # 66886-U)
with an 18-gauge needle and mounted in 200 μL tips (Eppendorf),
as shown in Supporting Methods. For StageTip
SPE processing using the Spin96, StageTips were inserted into a holder
and placed in the top, which was then stacked onto the wash-bottom
containing a polypropylene 96-well microtiter plate to collect the
sample flow-through and washes (Figure S1a). Each tip was wetted with 100 μL of 100% acetonitrile and
centrifuged at 1000g for 1 min. Following wetting,
each StageTip was equilibrated with 100 μL of 0.1% TFA in H2O
and 30% methanol/1% TFA with centrifugation for each at 1000g for 3 min. Each StageTip was then loaded with the equivalent
of ∼10 μg peptide in 49.5% ethyl acetate and 0.5% TFA
(equal volumes of each phase used). The peptides were washed twice
with 100 μL of 99% ethyl acetate/1% TFA, which was followed
by one wash with 100 μL of 0.2% TFA in water. For elution of
peptides, the wash-bottom was exchanged with a bottom containing a
holder supporting an unskirted PCR plate that has been trimmed to
fit (Figure S1b). To elute, 100 μL
of 5% ammonium hydroxide/80% acetonitrile was added to each tip and
centrifuged as above for 5 min. Samples in the PCR plate were dried
using a GeneVac EZ-2 using the ammonia setting at 40 °C for 1
h. Dried peptides were resuspended in 30 μL of 5% formic acid
and a CBQCA assay (see below) was performed to ensure even sample
loading. Samples were then stored at 4 °C until analyzed by LC–MS.