Figure S1. Correlation between the three score vectors identified from the PCA model of UV spectra and the concentration profiles of either the
individual protein or total protein in mixtures. Correlation coefficients with p-values less than 0.05 and 0.01 are represented with * and **,
respectively.
BSA (mg/ml)
6
first PC
4
BLG (mg/ml)
6
r = 0.50 **
4
4
r = 0.72 **
2
2
0
0
0
0
-2
-2
-2
-2
-4
-4
-4
-4
-6
0.3
-6
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
-6
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
0.5
0.3
0.3
0.2
0.2
0.1
0.1
0.1
0.1
0.0
0.0
0.0
0.0
-0.1
-0.1
-0.1
-0.1
-0.2
-0.2
-0.2
-0.2
0.2
r = 0.32 *
-0.3
-0.3
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
0.4
0.3
r = -0.83 **
0.4
r = - 0.07
0.3
r = 0.27
0.3
0.5
r = -0.09
0.2
0.1
0.1
0.1
0.1
0.0
0.0
0.0
0.0
-0.1
-0.1
-0.1
-0.1
-0.2
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
1.0
3.0
3.5
1.5
2.0
2.5
3.0
3.5
2.5
3.0
3.5
r = 0.06
0.3
0.2
-0.2
2.5
0.4
0.2
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
2.0
-0.3
0.2
-0.2
1.5
r = -0.14
0.2
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
0.4
r = 0.25
1.0
0.3
-0.3
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
r = 0.95 **
4
2
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
second PC
r = 0.42 **
total proteins (mg/ml)
6
2
-6
third PC
IgG (mg/ml)
6
-0.2
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
0.5
1.0
1.5
2.0
Figure S2. The actual vs. estimated protein concentrations obtained from the MCR-ALS model; (a) BSA (b) BLG (c) IgG. MCR-ALS model was
developed from the calibration set, and validated by external test set.
(b)
1.4
(c)
1.4
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.2
0.4
0.6
0.8
1.0
true BSA (mg/ml)
1.2
1.4
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.0
1.8
1.6
1.2
estimated IgG (mg/ml)
1.2
estimated BLG (mg/ml)
estimated BSA (mg/ml)
(a)
0.0
0.0
0.2
0.4
0.6
0.8
1.0
true BLG (mg/ml)
calibration
test
1.2
1.4
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
true IgG (mg/ml)
Figure S3. The VIP scores obtained from different PLS models. (a) 0th order + mean-centering (b) 1st
order + mean-centering (c) 2nd order + mean-centering (d) 3rd order + mean-centering (e) 4th order +
mean-centering (f) 0th order + auto-scaling (g) 1st order + auto-scaling (h) 2nd order + auto-scaling (i) 3rd
order + auto-scaling (j) 4th order + auto-scaling. Red line: BSA, blue line: BLG, green line: IgG.
VIP
(b)
(c)
(f)
5
3
3
VIP
4
2
1
0
250 260 270 280 290 300 310 320
0
250 260 270 280 290 300 310 320
wavelength (nm)
6
(g)
5
4
4
3
2
1
1
0
250 260 270 280 290 300 310 320
0
250 260 270 280 290 300 310 320
wavelength (nm)
wavelength (nm)
(h)
5
8
6
3
VIP
VIP
2
4
1
2
0
250 260 270 280 290 300 310 320
0
250 260 270 280 290 300 310 320
wavelength (nm)
wavelength (nm)
(i)
4
8
6
VIP
VIP
3
2
2
4
1
2
0
250 260 270 280 290 300 310 320
0
250 260 270 280 290 300 310 320
wavelength (nm)
4
(j)
wavelength (nm)
5
4
VIP
3
VIP
wavelength (nm)
6
5
3
(e)
2
1
4
(d)
5
4
VIP
VIP
(a)
2
1
3
2
1
0
250 260 270 280 290 300 310 320
0
250 260 270 280 290 300 310 320
wavelength (nm)
wavelength (nm)
Figure S4. The permutation test for PLS models. (a) BSA (b) BLG (c) IgG. During the permutation tests, 50 sets of the randomly permuted
Y were created, and their Q2 values were computed for the models with and without permutations to check the validity of Q2 obtained
from the original PLS model. The general guideline is that Q2-intercepts should not exceed 0.05 for the usable and valid PLS models.
(a)
(b)
1
(c)
1
1
Y = 1.7 * X - 0.8
Y = 2.5 * X - 1.6
Y = 1.6 * X - 0.7
0
0
0
-1
Q2
Q2
Q2
-1
-2
-1
-2
-3
-3
-4
0.0
0.2
0.4
0.6
0.8
1.0
correlation coefficients, r2
-2
0.0
0.2
0.4
0.6
0.8
1.0
correlation coefficients, r2
0.0
0.2
0.4
0.6
0.8
1.0
correlation coefficients, r2