the coupling was successful, check the MFI. Depending on the
dilution of the fluorophore, the signals can vary between 5000
and 10,000 for a good coupling.
9. We have found that thawing overnight at +4
C was most
practical.
10. Do not interrupt the experimental flow after dissolving
NHS-biotin, as this active substance is susceptible to hydrolysis
resulting in a loss in activity. Add NHS-biotin to the side of
each well using single- or multichannel dispensers so that the
labeling reactions for all samples are started simultaneously.
11. Every hour, take out the plate from the ice, vortex, and centrifuge it (500 Â g, 1 min).
12. Thaw the samples on ice the day of the assay run.
13. We have observed that heat treatment of labeled samples in
combination with the applied multiplexed assay procedure
allowed to modulate antibody performance [7]. This can lead
to improved protein detectability by changing the accessibility
of the epitopes in the complex sample solution but should be
tested and balanced with the tendency of proteins to precipitate
at higher temperatures.
Acknowledgments
The Proteomics Unit belongs to ProteoRed, PRB2- ISCIII
(PT17/0019/0014). I.R.P is supported by the Contrato Miguel
Servet-II FIS (contract CPII17/00026). L.L. is supported by the
Instituto de Salud Carlos III (ISCIII)Xunta de Galicia through a
postdoctoral fellowship Sara Borrell (CD19/0019)(IN606B2016/2005). This work was also supported by the KTH Center
for Applied Precision Medicine funded by the Ehrling Persson
foundation, as well as the Human Protein Atlas project funded by
Knut and Alice Wallenberg Foundation.
References
1. Lourido L, Ayoglu B, Ferna ´ndez-Tajes J,
Oreiro N, Henjes F, Hellstro ¨m C, Schwenk
JM, Ruiz-Romero C, Nilsson P, Blanco FJ
(2017) Discovery of circulating proteins associated to knee radiographic osteoarthritis. Sci
Rep
7(1):137.
https://doi.org/10.1038/
s41598-017-00195-8
2. Ayoglu B, Chaouch A, Lochmu ¨ller H,
Politano L, Bertini E, Spitali P, Hiller M, Niks
EH, Gualandi F, Ponte ´n F, Bushby K, AartsmaRus A, Schwartz E, Le Priol Y, Straub V,
Uhle ´n M, Cirak S, ‘t Hoen PA, Muntoni F,
Ferlini A, Schwenk JM, Nilsson P, Al-Khalili
Szigyarto C (2014) Affinity proteomics within
rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies. EMBO Mol
Med
6(7):918–936.
https://doi.org/10.
15252/emmm.201303724
3. H€ aggmark A, Mikus M, Mohsenchian A, Hong
MG, Forsstro ¨m B, Gajewska B, Baran ´ czykKuz ´ma A, Uhle ´n M, Schwenk JM, Kuz ´maKozakiewicz M, Nilsson P (2014) Plasma
profiling reveals three proteins associated to
amyotrophic lateral sclerosis. Ann Clin Transl
150
Lucı ´a Lourido et al.
dilution of the fluorophore, the signals can vary between 5000
and 10,000 for a good coupling.
9. We have found that thawing overnight at +4
C was most
practical.
10. Do not interrupt the experimental flow after dissolving
NHS-biotin, as this active substance is susceptible to hydrolysis
resulting in a loss in activity. Add NHS-biotin to the side of
each well using single- or multichannel dispensers so that the
labeling reactions for all samples are started simultaneously.
11. Every hour, take out the plate from the ice, vortex, and centrifuge it (500 Â g, 1 min).
12. Thaw the samples on ice the day of the assay run.
13. We have observed that heat treatment of labeled samples in
combination with the applied multiplexed assay procedure
allowed to modulate antibody performance [7]. This can lead
to improved protein detectability by changing the accessibility
of the epitopes in the complex sample solution but should be
tested and balanced with the tendency of proteins to precipitate
at higher temperatures.
Acknowledgments
The Proteomics Unit belongs to ProteoRed, PRB2- ISCIII
(PT17/0019/0014). I.R.P is supported by the Contrato Miguel
Servet-II FIS (contract CPII17/00026). L.L. is supported by the
Instituto de Salud Carlos III (ISCIII)Xunta de Galicia through a
postdoctoral fellowship Sara Borrell (CD19/0019)(IN606B2016/2005). This work was also supported by the KTH Center
for Applied Precision Medicine funded by the Ehrling Persson
foundation, as well as the Human Protein Atlas project funded by
Knut and Alice Wallenberg Foundation.
References
1. Lourido L, Ayoglu B, Ferna ´ndez-Tajes J,
Oreiro N, Henjes F, Hellstro ¨m C, Schwenk
JM, Ruiz-Romero C, Nilsson P, Blanco FJ
(2017) Discovery of circulating proteins associated to knee radiographic osteoarthritis. Sci
Rep
7(1):137.
https://doi.org/10.1038/
s41598-017-00195-8
2. Ayoglu B, Chaouch A, Lochmu ¨ller H,
Politano L, Bertini E, Spitali P, Hiller M, Niks
EH, Gualandi F, Ponte ´n F, Bushby K, AartsmaRus A, Schwartz E, Le Priol Y, Straub V,
Uhle ´n M, Cirak S, ‘t Hoen PA, Muntoni F,
Ferlini A, Schwenk JM, Nilsson P, Al-Khalili
Szigyarto C (2014) Affinity proteomics within
rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies. EMBO Mol
Med
6(7):918–936.
https://doi.org/10.
15252/emmm.201303724
3. H€ aggmark A, Mikus M, Mohsenchian A, Hong
MG, Forsstro ¨m B, Gajewska B, Baran ´ czykKuz ´ma A, Uhle ´n M, Schwenk JM, Kuz ´maKozakiewicz M, Nilsson P (2014) Plasma
profiling reveals three proteins associated to
amyotrophic lateral sclerosis. Ann Clin Transl
150
Lucı ´a Lourido et al.