SUPPORTING INFORMATION
Samples and chemicals
The test sample analyzed was a mixture of diluted, but otherwise untreated, human plasma obtained from a healthy volunteer.
RapiGest SF (lyophilized sodium-3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxyl]-
1-propane-sulfonate) was obtained from Waters (Milford, MA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Proteomics grade trypsin (also Sigma-
Aldrich) was used in all digestions.
In-solution digestion prior to enrichment
To 125 μL sample (containing 2.5 μL plasma in water) 3 μL 0.5% RapiGest SF and 2 μL 200 mM 1,4-dithioD -threitol (DTT) were added and the mixture was kept at 60 °C for 30 min to achieve protein unfolding and reduction. Following reduction, alkylation was performed by adding 15 μL 200 mM NH
4
HCO
3
and 2 μL 200 mM 2-iodoacetamide (IAA) and storing the sample for 30 min in the dark, at room temperature. The alkylated samples were digested at
37 °C for 180 min with trypsin (10 μL, 40 μM), followed by microwave irradiation twice for 5 min. The digestion was quenched by adding1 μL formic acid (30 min at 37 °C); which also served to degrade the surfactant and to phase-separate the hydrophobic part from the digested sample. The reaction product was centrifuged at 13500 rpm (corresponding to 17000 g ) for 10 min, and enrichment was then performed on 100 μL of supernatant.
In-solution digestion after enrichment

Each fraction was concentrated by a SpeedVac. In the case of fractions containing acidic media solvent exchange was performed in VivaSpin centrifuge tubes (Sartorius AG, Goettingen,
Germany) by washing the samples with water three times. The end volume of each fraction was
100 µL. From each fraction 10 µL was digested the following way:
1.5 μL 0.5% RapiGest SF and 1 μL 200 mM DTT was added and kept at 60 °C for 30 min to achieve protein unfolding and reduction. Following reduction 2.5μL 200 mM NH
4
HCO
3
and 1.5
μL 200 mM IAA were added to alkylate the sample for 30 min in the dark, at room temperature.
The alkylated samples were digested at 37 °C for 180 min with trypsin (2 μL, 4 μM), followed by microwave irradiation twice for 5 min. The digestion was quenched by adding1 μL formic acid (30min at 37 °C); which also served to degrade the surfactant and to phase-separate the hydrophobic part from the digested sample. The reaction product was centrifuged at 13500 rpm
(corresponding to 17000 g ) for 10 min, and enrichment was then performed.
Lectin affinity chromatography
Lectin affinity chromatography was performed on an AffiSep-WGA-SPE cartridge (GALAB
Technologies GmbH, Geesthacht, Germany) (0.6 mL/4.0 × 50 mm). First, the cartridge was conditioned using 3600 μL binding buffer (10 mM BIS-TRIS pH = 6.0, 15 mM NaCl, 1 mM
CaCl
2
, 1 mM MnCl
2
). Ten times diluted blood plasma sample in binding buffer or digested supernatant was applied on the cartridge (fraction 1). The cartridge was washed with 1200 μL binding buffer (fraction 2). Elution was performed by adding 1200 μL elution buffer (10 mM
BIS-TRIS pH = 6.0, 15 mM NaCl, 1 mM CaCl
2
, 1 mM MnCl
2
, 150 mM N -acetylglucosamine) and the elute was collected (fraction 3).

Phenylboronic acid (PBA) affinity chromatography
PBA cartridges (BondElut PBA, Agilent Technologies, Santa Clara, CA, USA) were pretreated with 400 μL of 90% acetonitrile, 5% formic acid. The cartridges were washed with 1 mL of 200 mM ammonium hydrogen carbonate buffer (pH 10). Ten times diluted blood plasma sample in
200 mM ammonium hydrogen carbonate buffer (pH 10) or digested supernatant was applied on the cartridge (fraction 1). The unbound proteins/peptides were washed with 400 μL 200 mM
NH
4
HCO
3
pH 10 (fraction 2) and then with 600 μL 75% ACN + 25% 200 mM NH
4
HCO
3
pH 10
(fraction 2). Elution was performed by adding 800 μL 50% ACN 5% FA and the elute was collected (fraction 3).
Pre-equilibrated anion-exchange SPE
ISOLUTE PE-AX SPE cartridges (25 mg sorbent/1 mL SPE; Biotage AB, Uppsala, Sweden) were conditioned with 400 μL 99% ACN, 20 mM ammonium acetate. Ten times diluted blood plasma sample in 20 mM ammonium acetate or 100 µL digested supernatant was applied on the cartridge and washed with 200 μL 10% ACN, 50 mM ammonium acetate (fraction 1). Elution was performed by adding 300 μL 10% ACN, 5% FA (fraction 2) and then with 300 μL 10%
ACN, 300 mM citric acid (fraction 3).
LC/MS(MS) measurements
LC/MS(MS) analysis of the tryptic peptide mixtures was performed using a nanoflow UPLC system (nanoAcquity UPLC, Waters) coupled to a high-resolution quadrupole-time-of-flight
(QTOF) Premier mass spectrometer (Waters). The electrospray emitter was purchased from New
Objective, Woburn, MA, USA. The tryptic peptides were desalted online on a Waters Symmetry

C18 trap column (180 µm i.d. × 20 mm) and then separated on a Waters reversed-phase analytical column (C18, 75 µm i.d. × 200 mm, 1.7 µm BEH particles). The elution of the peptides from the analytical column to the emitter tip was achieved using a flow rate of 400 nL/min and a 70 min long gradient going from 10% to 50% solvent B (solvent A was water containing 0.1% formic acid and solvent B was acetonitrile also containing 0.1% formic acid).
Electrospray ionization mass spectrometry was used in the positive ion mode. Signal intensities were measured using single-stage mass spectrometry (cone voltage: 15, 1 eV collision energy, capillary voltage 2.7 kV, nebulizing gas (N
2
) pressure 0.5 bar, cone gas (N
2
) flow rate 15
L/h, source temperature: 90 °C). Protein identification was performed using tandem mass spectrometry in the survey mode (DDA survey) from m/z 400 to 1999. The collision gas was argon, at 4 × 10
–3
mbar pressure. The collision energy was varied in the 12–70 eV range, according to predefined charge-state-dependent configuration files.