Received: 22 July 2021 Revised: 22 September 2021 Accepted: 19 October 2021

DOI: 10.1002/jpen.2285

O R I G I N A L C O M M U N I C AT I O N

Proteomics analysis reveals digestion-resistant proteins from

colostrum are associated with inflammatory and cytotoxic
responses in intestinal epithelial cells
Yimin Chen PhD, RDN Assistant Professor1,2 Bum Jin Kim PhD Postdoctoral Scholar3
David C. Dallas PhD Assistant Professor3

1
Department of Kinesiology and Nutrition,
University of Illinois at Chicago, Chicago, Abstract
Illinois, USA
Background: Although human-milk feeding reduces the risk of necrotizing ente-
2
Margaret Ritchie School of Family and
Consumer Sciences, University of Idaho,
rocolitis (NEC) in preterm infants compared with formula feeding, the exact risk-
Moscow, Idaho, USA reduction mechanism remains unknown. As NEC occurs at the distal small intestine
3
Oregon State University, Corvallis, Oregon, in which digestion has occurred, we applied proteomics to examine the extent to which
USA
colostrum proteins survive simulated infant in vitro–digestion and, thus, have potential
Correspondence to exert biological function.
Yimin Chen, University of Idaho, 875 Perimeter
Methods: Ten preterm colostrum samples were left undigested or in vitro–digested,
Dr, MS 3183, Moscow, ID, USA.
Email: yiminc@uidaho.edu and lipopolysaccharide (LPS)-binding protein, soluble cluster of differentiation 14, and
tumor necrosis factor (TNF) receptors I and II were measured using enzyme-linked
Statistical consultant: Yanming Di, PhD, Ore-
gon State University. immunosorbent assay in all undigested and in vitro–digested samples. Fully differen-
tiated Caco-2 cells were exposed to digested colostrum samples before stimulation
Funding information
USDA Multistate Workgroup, Grant/Award
with LPS or TNF or no stimulation. Inflammation (interleukin-8) and cytotoxicity (lac-
Numbers: W4002, K99/R00; Pathway to Inde- tate dehydrogenase) were measured. Proteomic analyses of undigested and in vitro–
pendence Career Award, Eunice Kennedy
Shriver Institute of Child Health & Devel-
digested samples were done using mass spectrometry.
opment of the National Institutes of Health, Results: We found that most proteins in colostrum are significantly, if not com-
Grant/Award Number: R00HD079561; USDA
National Institute of Food and Agriculture,
pletely, degraded after in vitro–digestion. We found select individual and combina-
Grant/Award Number: 2018-67017-27521; tion digestion-resistant proteins that were positively correlated with LPS- and TNF-
Gerber Foundation, Grant/Award Number:
2017-1586; International Society for Research
induced inflammation.
on Human Milk and Lactation–Family Larsson- Conclusion: These results indicate the importance of considering the extent to which
Rosenquist Foundation Trainee Expansion
Program
specific dietary compounds survive digestion to reach their site of claimed action (distal
intestine) and that some digestion-resistant proteins may be contributing toward “low-
grade” inflammation that is necessary to promote intestinal growth and maturation
during early infancy. This work provides the most detailed understanding of human-
milk protein degradation with simulated infant in vitro–digestion to date.

KEYWORDS
gastroenterology, immunonutrition, life cycle, neonates, research and diseases

© 2021 American Society for Parenteral and Enteral Nutrition

JPEN J Parenter Enteral Nutr. 2021;1–11. wileyonlinelibrary.com/journal/jpen 1

2 CHEN ET AL
CLINICAL RELEVANCY STATEMENT
Human-milk feeding reduces risk for necrotizing enterocolitis in
preterm infants, which is commonly located in the distal small intestine,
in which full digestion has occurred. Through proteomics, we observed
that some digestion-resistant human-milk proteins contribute toward
low-grade inflammation that is necessary to promote intestinal growth
and maturation.
INTRODUCTION
Human-milk (HM) feeding decreases risk for necrotizing enterocolitis
(NEC), a severe inflammatory bowel disease, in premature infants.1,2
Intact HM contains many immunomodulatory proteins (eg, cytokines
and growth factors) that ameliorate intestinal inflammation in infants.3
Colostrum, HM produced during the first few days of lactation, con-
tains the highest concentration of immunomodulatory proteins (eg,
secretory IgA and lactoferrin) in comparison with later stages of
lactation.4,5
However, NEC occurs in the distal small intestine. After entering the
digestive system, milk proteins are exposed to changes in pH and an
array of digestive proteases that can degrade many proteins into pep-
tides and amino acids. Although preterm infants have reduced ability to
digest proteins compared with term infants, there is evidence that pro-
teins are digested to some degree.6–9 The extent to which specific pro-
teins in HM can modulate the inflammatory cascade, and thus reduce
NEC risk, depends on their structural survival across digestion to the
distal small intestine. However, there are little data on how preterm-
infant digestion alters the presence, form, and bioactivities of specific
colostrum proteins.
We previously demonstrated that in vitro–digested colostrum
and transitional HM (days 2–8 postpartum) significantly suppress
lipopolysaccharide (LPS)/tumor necrosis factor (TNF)–induced inflam-
mation and cytotoxicity in intestinal epithelial cells, with significant
variability between different mothers’ milks.10 The causative factor for
why only the digested samples had this effect and why such large vari-
ation in effect was observed remained unknown. As a major change
that occurs with in vitro digestion is the partial breakdown of proteins,
we hypothesized that changes in the intact protein profile (losses of
certain proteins and gains of certain peptides) are associated with the
antiinflammatory and anticytotoxic effects and vary between mothers’
milks.
MATERIALS AND METHODS
Colostrum samples
Ten deidentified colostrum samples from different mothers of prema-
ture infants were obtained from an existing HM repository, and each
were separated into two aliquots: (1) undigested and (2) digested using
an in vitro–digestion protocol to simulate premature-infant diges-
tion, as described previously.10,16 In brief, colostrum samples (200
μl colostrum + 2.2235 ml deionized water) designated as “digested”
were treated with pepsin (80 mg pepsin [≥250 units/mg] in 2 ml of
deionized water) and titrated to pH 4.0 with 1 M hydrochloric acid
(gastric digestion) and kept at 37 ◦ C with 100 rpm shaking for 30 min.
A variable amount (10–22 μl) of 1 M sodium bicarbonate was added
to increase pH to 6.0 and pancreatin and bile salts (20 mg pancreatin
[4X U.S. Pharmacopeia (USP)] + 120 mg bile salt in 10 ml .1 M sodium
bicarbonate) were added, followed by incubation at 37 ◦ C with 100
rpm shaking for 30 min and then pH was adjusted to 7.0, followed by
incubation for 2 h at 37 ◦ C with 100 rpm shaking to mimic intestinal
digestion.10,11 All samples were heat treated at 90 ◦ C for 15 min
to deactivate enzymatic reactions as the final step. All undigested
colostrum samples were diluted with deionized water to the same
final volume as their matched digested samples and exposed to the
same final heat treatment. All undigested and digested colostrum
samples were either kept as is or skimmed: in HM protein studies, the
fat portion is commonly removed to isolate and measure the protein
contents in skimmed portion of HM only. However, protein content in
the fat layer can contain up to 4% of total HM proteins, as previously
demonstrated in the literature.12 Thus, it is important to compare
protein concentrations measured in both skimmed and unskimmed
HM samples to determine the amount of contribution from the fat
layer towards the total measured content of target proteins. All undi-
gested, digested, skimmed, and unskimmed colostrum samples were
stored at −80 ◦ C until use for cell culture experiments and proteomic
analyses.
Intestinal Epithelial Cell Cultures
Caco-2BBe cells were grown at 37 ◦ C with 5% carbon dioxoide in Dul-
becco’s Modified Eagle Medium (DMEM, Gibco, NY,) containing 10%
heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, and
1% nonessential amino acids (Gibco, NY) on type I collagen (Sigma-
Aldrich, St Louis, MO)–coated 48-well plates. Cells were grown for 21
days after confluence to obtain fully differentiated cells. Passages 23
and 24 were used for experiments.
Experimental Conditions
Caco-2BBe cell monolayers were treated with 1 of 10 in vitro–
digested colostrum at 2% (vol/vol in DMEM) or no pretreatment
(control: continue incubation with DMEM to maintain usual cell
growth) for 1 h at 37 ◦ C. The cells were then stimulated by adding
10 μl of either 100 μg/ml LPS, from Escherichia coli 0111:B4, Sigma,
St Louis, MO), or 100 ng/ml recombinant human TNF (PeproTech,
Rocky Hill, NJ) or left unstimulated (10 μL of culture medium) and
incubated overnight. All experimental conditions were performed in
triplicate.
JOURNAL OF PARENTERAL AND ENTERAL NUTRITION
Inflammation and cytotoxicity measurements
Following overnight incubation, supernatants were collected from
each well. Interleukin 8 (IL-8) (marker of inflammation) were measured
using enzyme-linked immunosorbent assay (ELISA) (R&D Systems,
Minneapolis, MN). The lactate dehydrogenase (LDH) assay (Pierce LDH
Cytotoxicity Assay Kit, Pierce Biotechnology, Rockford, IL) was used to
determine the cytotoxic effect of the digested colostrum. For this assay,
three additional wells of cells were maintained overnight without pre-
treatment or stimulation. On the following morning during supernatant
extraction, lysis buffer was added to these three control wells for 45
min to create a maximum LDH–release measurement for calculations.
All samples, controls, and maximum LDH–release wells were then mea-
sured for LDH activity. Percentage of cytotoxicity for each sample was
then calculated as (treated samples LDH − control LDH)/(maximum
LDH − control LDH) × 100.
Protein quantification
LPS-binding protein (LBP), soluble cluster of differentiation 14
(sCD14), and TNF receptor I (TNFRI) and TNF receptor II (TNFRII)
(molecules that interact directly with the LPS and TNF stimuli used in
the cell culture experiments) were measured by ELISA (LBP and TNFRI
[DuoSet R&D Systems]; sCD14 and TNFRII [Quantikine R&D Systems])
in all samples in duplicate.
Colostrum protein extraction
All undigested and digested colostrum samples (500 μl) were cen-
trifuged at 1800 x g for 20 min at 4 ◦ C to separate the lipid layer. The
infranatant was removed using extended gel-loading pipette. Ethanol
precipitation was performed as previously described,13 with the fol-
lowing changes. Two hundred microliters of ethanol were added to
50 μl of each sample and protein pellet was collected. To compare
the efficiency of protein extraction methods, trichloroacetic acid (TCA)
precipitation and 10-kDa molecular weight cutoff (MWCO)–based
centrifugal filtration were also examined using a single colostrum sam-
ple. TCA precipitation and centrifugal filtration were conducted as in
our previous study.14 The ethanol precipitation method yielded the
highest number of tryptic peptides identified compared with MWCO-
based filtration and TCA precipitation (Figure S1); thus, ethanol precip-
itation was used for this study.
Total protein concentration measurement
Total protein concentration was measured using the bicinchoninic acid
(BCA) protein assay (Pierce, Rockford, IL) in ethanol-precipitated pro-
tein pellets (protein pellets were dissolved in ultrapure water followed
by a minimum 1-min vortex, per manufacturer recommendation) from
all undigested and in vitro–digested colostrum samples. The total pro-
3
tein concentrations were measured to calculate percentage of protein
degradation as a point of validation against proteomics data. Percent-
age of protein degradation for each colostrum sample was calculated
as (protein concentration of undigested sample – protein concentra-
tion of digested sample)/protein concentration of undigested sample ×
100.
Solid phase extraction
After protein extraction, the protein pellets were dissolved and
digested with trypsin. Solid phase extraction of the trypsin-digested
colostrum samples was conducted using C18 96-well plates (Glygen
Corp, Columbia, MD). Each C18 well was first activated using 200 μl
of 99% acetonitrile (ACN) in .1% trifluoroacetic acid (TFA) three times,
then rinsed with 200 μl of 1% ACN in .1% TFA three times, followed by
loading 200 μl of sample and three subsequent rinses with 1% ACN in
.1% TFA as above, and finally, each well was eluted with 200 μl of 80%
ACN in .1% TFA three times. Between each step, the plates were cen-
trifuged at 1000 rpm for 1 min at 4 ◦ C. The eluted samples were col-
lected and dried by freeze dryer overnight. Samples were rehydrated
in 50 μl of 3% ACN prior to MS analysis.
Liquid chromatography-nanoelectrospray ionization
mass spectrometry
Peptides were measured using a Waters nanoACQUITY ultraperfor-
mance liquid chromatography (UPLC) (Waters, Milford, MA) with Orbi-
trap Fusion Lumos Tribrid mass spectrometry (MS) (Thermo Scien-
tific, Waltham, MA). One microliter of peptides were loaded onto a
C18 180 μm × 20 mm, 5-μm bead nanoAcquity UPLC trap column
(Waters) for enrichment and desalting and separated with a 100 μm
× 100 mm, 1.7-μm bead Acquity UPLC Peptide BEH C18 column
(Waters) over 60 min. Peptides were separated at a flowrate of .5
μl/min with a gradient using the following proportions and time points:
3%–11.5% B, 0–10 min; 11.5%–20% B, 10–31 min; 20%–30% B, 32–
36 min; 30%–95% B, 36–45 min; 95% B, 45–54.5 min, 95−3% B over
.5 min, and then finally the column was reequilibrated with 97% A
for 5 min. Solvent A was 100% ultrapure water with .1% formic acid,
and solvent B was 100% ACN with .1% formic acid. Peptides were
ionized with an electrospray voltage of 2320 V and an ion trans-
fer tube temperature of 300 ◦ C. MS spectra were acquired in posi-
tive ionization mode over an m/z (mass-to-charge) range of 300–2000
with a resolution of 60,000. The automatic gain control (AGC) target
was set to 4.0 × 105 , with a maximum injection time of 50 ms. The
MS cycle time was set to 3 s. Following an MS scan, precursor com-
pounds were automatically selected for MS/MS analysis by the acqui-
sition software based on the following criteria: most intense peaks,
ion-intensity threshold 5.0 × 104 , and charge state 2–8. The frag-
mentation mode was set to electron-transfer/higher-energy collision
dissociation (EThcD), with data-dependent analysis. EThcD was per-
formed with optimized electron-transfer dissociation reaction times
4 CHEN ET AL
depending on charge state (2+, 130 ms; 3+, 70 ms; 4+, 50 ms; 5+, 40
ms; and 6+ to 8+, 20 ms) and supplemental higher-energy collision-
induced dissociation activation (25% of collision energy). MS/MS spec-
tra were acquired in the positive ionization mode over an m/z range of
300–2000 by the Orbitrap at resolution of 30,000. The AGC target was
set to 5.0 × 104 for the EThcD method.
MS data analysis
Raw data files for each sample were analyzed using Thermo Proteome
Discoverer (v2.2), with a SequestHT search engine to identify peptides
using an in-house HM protein sequence database. Peptide sequences
of all proteins identified in digested colostrum samples that were not
identified in undigested colostrum were examined to rule out porcine
origin (porcine-derived digestive enzymes were used for the in vitro–
digestion protocol) using the Basic Local Alignment Search Tool protein
of the National Library of Medicine.
Statistical analyses
All data were analyzed using SPSS (IBM Corp, 2015, Version 23.0;
Armonk, NY). Wilcoxon signed rank test was used to compare the dif-
ferences in IL-8 and percentage of cytotoxicity between Caco-2BBe
cells with and without either LPS or TNF stimulation. Multivariate anal-
ysis of variance (MANOVA) (general linear model with multivariate
testing for between-samples factors) was then performed to deter-
mine IL-8 and percentage of cytotoxicity differences both between
with and without either LPS or TNF stimulation and among different
digested colostrum samples. Paired t-tests were used to compare pro-
tein differences between undigested and digested samples. All cell cul-
ture data and MS protein abundance data were then log transformed
for the remainder of analyses because of data ranging over several
orders of magnitude. Linear regressions were used to detect corre-
lations between each protein found in in vitro–digested samples and
the outcomes: IL-8 and percentage of cytotoxicity reduction. Multi-
ple regression was then used to identify correlations between groups
of proteins found in in vitro–digested samples and the outcomes: IL-8
and percentage of cytotoxicity reduction. For heat-map generation, all
protein data were first normalized to the most abundant value within
each protein across all samples. Proteins were then categorized based
on primary bioactivity: (1) immune, (2) transport, (3) enzyme, or (4)
cell/protein regulation.
RESULTS
Intestinal epithelial cell outcomes
LPS stimulation significantly increased IL-8 production from intesti-
nal epithelial cells compared with no stimulation (152.9 (interquartile
range [IQR] 130.8–189.6] vs 69.4 [IQR 46.3–98.5]; P < .001). TNF stim-
ulation significantly increased both IL-8 production (402.8 [IQR 164.1–
587.7] vs 69.4 [IQR 46.3–98.5]; P < .001) and percentage of cytotoxic-
ity (21.0 [IQR 15.0–26.8] vs 9.6 [IQR −3.0–31.7]; P = .003) compared
with no stimulation. Treatment with digested colostrum samples as an
aggregate did not significantly reduce IL-8 production and percentage
of cytotoxicity. However, MANOVA analyses showed significant differ-
ences in the effects of digested colostrum samples on reducing IL-8
production (P = .006) and percentage of cytotoxicity (P < .001). Differ-
ent digested colostrum samples exerted significantly different reduc-
tion on IL-8 production under no stimulation (P = .016) and TNF stimu-
lation (P = .027), as well as cytotoxicity under no stimulation P = .015)
and LPS stimulation (P = .048).
Selected protein measurements using ELISA
All measured proteins (LBP, sCD14, TNFRI, and TNFRII) decreased
significantly (P < .001) after in vitro digestion compared with undi-
gested colostrum samples (Table 1). Skimming of both undigested and
in vitro–digested colostrum samples did not significantly alter pro-
tein concentrations. After in vitro digestion, all proteins fell below
detection limits in all nonskimmed and skimmed colostrum samples
(Table 1).
Total protein concentrations
Based on BCA assay measurements, average total intact (ethanol pre-
cipitable) protein concentration significantly decreased when com-
paring undigested vs in vitro–digested colostrum samples (2434 ±
442 mcg/ml vs 529 ± 240 mcg/ml; P < .001). The mean percentage of
protein degradation was 82%. Percentage of total protein degradation
itself was not correlated with IL-8 and cytotoxicity reductions.
Protein identification and degradation
Peptides derived from their intact HM proteins via trypsin digestion
were enriched and separated online by a chromatographic method.
Subsequent MS profiling enabled identification and differentiation of
peptides by highly accurate MS analysis and interpretation of tandem
MS spectra. Determination of the abundance of the intact proteins was
accomplished by assigning peptide sequences and their abundances
to proteins. A total of 203 proteins were identified in the 10 undi-
gested colostrum samples. Of these, 69 were also identified in the in
vitro–digested colostrum samples (Table S1); thus, 66% of all identified
proteins from undigested colostrum samples were degraded beyond
detection limits (Figure 1). Total protein-assigned ion abundance was
significantly lower in digested colostrum samples compared with undi-
gested samples (Figure 2). Of the 69 proteins that were retained in the
in vitro–digested colostrum samples, 44 were >90% degraded based on
relative abundance, including many commonly identified HM proteins
(selected proteins in Table 2). Four proteins (TNF receptor–associated
JOURNAL OF PARENTERAL AND ENTERAL NUTRITION 5

TA B L E 1 Protein mediator concentrations in colostrum samples with/without in vitro digestion and skimming

LBP (ng/ml) sCD14 (pg/ml) TNFRI (pg/ml) TNFRII (pg/m;)

Colostrum 1
Undigested <.781 14,272.94 ± 252.49 448.90 ± 11.00 322.97 ± 1.60
Undigested skimmed <.781 14,384.14 ± 70.42 382.16 ± 2.05 335.90 ± 39.00
Digested <.781 <62.5 <12.5 <7.81
Digested skimmed <.781 <62.5 <12.5 <7.81
Colostrum 2
Undigested 2.96 ± .95 13,966.94 ± 924.59 570.47 ± 19.95 618.37 ± 7.83
Undigested skimmed <.781 14,555.71 ± 89.03 638.627 ± 3.74 627.80 ± 26.25
Digested <.781 <62.5 <12.5 <7.81
Digested skimmed <.781 <62.5 <12.5 <7.81
Colostrum 3
Undigested <.781 14,441.41 ± 229.51 421.22 ± 4.98 374.65 ± 17.65
Undigested skimmed <.781 14,660.98 ± 159.09 354.53 ± 10.67 405.79 ± 1.50
Digested <.781 <62.5 <12.5 <7.81
Digested skimmed <.781 <62.5 <12.5 <7.81
Colostrum 4
Undigested <.781 14,205.08 ± 241.91 259.28 ± 4.91 346.54 ± 4.62
Undigested skimmed <.781 14,432.38 ± 76.62 232.10 ± 1.32 305.43 ± 8.13
Digested <.781 <62.5 <12.5 <7.81
Digested skimmed <.781 <62.5 <12.5 <7.81
Colostrum 5
Undigested <.781 14,064.98 ± 569.94 501.05 ± 63.66 365.33 ± 4.22
Undigested skimmed <.781 14,175.15 ± 7.66 364.78 ± 21.83 433.91 ± 75.14
Digested <.781 <62.5 <12.5 <7.81
Digested skimmed <.781 <62.5 <12.5 <7.81
Colostrum 6
Undigested 19.05 ± 1.23 14,050.02 ± 456.09 >800 763.23 ± 43.69
Undigested skimmed 7.92 ± 2.76 14,327.89 ± 125.15 718.62 ± 11.15 769.67 ± 22.44
Digested <.781 <62.5 <12.5 <7.81
Digested skimmed <.781 <62.5 <12.5 <7.81
Colostrum 7
Undigested <.781 13,941.66 ± 873.51 354.87 ± 12.47 425.24 ± 5.27
Undigested skimmed <.781 14,371.49 ± 321.09 265.13 ± 20.41 463.80 ± 7.06
Digested <.781 <62.5 <12.5 <7.81
Digested skimmed <.781 <62.5 <12.5 <7.81
Colostrum 8
Undigested <.781 14,182.38 ± 572.49 298.43 ± 32.09 543.02 ± .65
Undigested skimmed <.781 14,437.80 ± 217.83 292.99 ± 18.74 531.62 ± 7.70
Digested <.781 <62.5 <12.5 <7.81
Digested skimmed <.781 <62.5 <12.5 <7.81
Colostrum 9
Undigested <.781 14,295.90 ± 431.65 477.44 ± 18.79 443.12 ± 2.70
Undigested skimmed <.781 14,446.57 ± 203.24 394.27 ± 13.15 431.94 ± 21.68
Digested <.781 <62.5 <12.5 <7.81
Digested skimmed <.781 <62.5 <12.5 <7.81

(Continues)
6 CHEN ET AL

TA B L E 1 (Continued)

LBP (ng/ml) sCD14 (pg/ml) TNFRI (pg/ml) TNFRII (pg/m;)

Colostrum 10
Undigested <.781 13,988.36 ± 398.08 412.53 ± 2.91 339.45 ± 3.79
Undigested skimmed <.781 14,395.49 ± 150.69 377.21 ± 30.20 355.86 ± 10.22
Digested <.781 <62.5 <12.5 <7.81
Digested skimmed <.781 <62.5 <12.5 <7.81

Note: All values within detection limits expressed with mean ± SD.
Abbreviations: LBP, lipopolysaccharide-binding protein; sCD14, soluble cluster of differentiation 14; TNFRI, tumor necrosis factor receptor I; TNFRII, tumor
necrosis factor receptor II.

factor 1, fibroblast growth factor, procathepsin H, and MUC5B) were

identified in some of the in vitro–digested colostrum samples that were
not present in the undigested samples.

High-abundance proteins in undigested colostrum

samples

The top 20 most abundant proteins, representing 87% of total protein-

assigned ion abundance in the undigested colostrum samples are listed
in Table 3. Lactoferrin was the most abundant protein (31.76% of total
protein-assigned ion abundance) followed by β-casein (17.41%), mak-
ing up nearly half of the proteins in undigested colostrum samples.

F I G U R E 1 Venn diagram demonstrating number of proteins

identified in 10 colostrum samples (total 203 proteins): 130 proteins
were identified only in undigested colostrum samples (gray area), 69
proteins were identified in both undigested and digested colostrum
samples (overlapped gray and pink areas), and four proteins were
identified only in digested colostrum samples (pink area)
High-abundant proteins in the in vitro–digested
colostrum samples
Among the 20 most abundant proteins in the in vitro–digested
colostrum samples, 10 were part of immunoglobulin protein com-
plexes, as shown in Table 4. Lactoferrin was the most abundant nonim-
munoglobulin protein. The remaining proteins in the top 20 consist of
mostly immune function–related proteins. The top 10 most abundant
proteins made up 95% of the total ion intensity ascribed to proteins in
the digested samples (Table 4). Although all casein proteins (β-casein,
κ-casein, and α-S1-casein) were >90% degraded, β-casein and κ-casein
remained in the top 10 most abundant proteins after in vitro digestion
(Table 4).

Comparison of proteins found in undigested and in

vitro–digested colostrum samples

Among the 69 proteins found both in undigested and in vitro–digested

F I G U R E 2 Total protein-assigned ion abundance between
undigested (n = 10) and digested (n = 10) colostrum samples.
*Protein-assigned ion abundances were significantly different
between undigested and digested colostrum samples using paired
t-test (P < .01)
colostrum samples, most proteins were significantly lower in ion
abundance in the in vitro–digested samples compared with the undi-
gested counterparts (P < .01). The exceptions were human leuko-
cyte antigen (HLA) class II histocompatibility antigen DR α chain, α-1-
antichymotrypsin, and 14-3-3 protein epsilon, which did not decrease
with digestion.
JOURNAL OF PARENTERAL AND ENTERAL NUTRITION 7

TA B L E 2 Abundance of select commonly known bioactive proteins (or associated proteins) in human milk in both undigested and in
vitro–digested matched colostrum samples and calculated percentage of protein degradation in digested vs undigested colostrum samples

Abundance in in
Abundance in undigested vitro–digested Degradation after in
Protein colostrum (n = 10) colostrum (n = 10) vitro digestion, %
Lactoferrin 32233236846 600658072 98.14
Polymeric immunoglobulin receptor 5589057139 442140074 92.09
Bile salt–activated lipase 1087329620 526718 99.95
Osteopontin 625379556 276978 99.96
Lysozyme 102515497 173608 99.83
Mucin-1 58316355 2068650 96.45
Mucin-4 22035794 1465468 93.35
Insulin-like growth factor–binding protein 2 4435613 0 100.00
Proepidermal growth factor 1817171 0 100.00
Lipopolysaccharide-binding protein 705592 0 100.00

TA B L E 3 Top 20 most abundant proteins in the undigested TA B L E 4 Top 20 most abundant proteins in the in vitro–digested
colostrum samples (n = 10) and their relative percentage of colostrum samples (n = 10) and their relative percentage of
concentration concentration

Percentage Percentage
of total of total
Protein Abundance protein Protein Abundance protein
Lactotransferrin 32233236846 31.76 Ig α-1 chain C region 1621351367 32.40
β-casein 17676352844 17.41 Ig κ chain C region 943612344 18.86
α-lactalbumin 12120043747 11.94 Lactotransferrin 600658072 12.00
Ig α-1 chain C region 6683853021 6.59 Polymeric Ig receptor 442140074 8.84
Polymeric immunoglobulin receptor 5589057139 5.51 Ig J chain 426226780 8.52
Serum albumin 5069970187 4.99 Ig α-2 chain C region 264472226 5.29
κ-casein 3212599585 3.17 Tenascin 120625048 2.41
Isoform 3 of α-S1-casein 2138765684 2.11 α-lactalbumin 118811220 2.37
Ig κ chain C region 1909867827 1.88 Complement C3 84967728 1.70
Tenascin 1775756544 1.75 Galectin-3 binding protein 69821367 1.40
Bile saltχactivated lipase 1087329620 1.07 Ig λ-2 chain C region 56215767 1.12
Clusterin 1070290924 1.05 Ig μ chain C region 36349205 .73
Immunoglobulin J chain 1040248332 1.02 Ig κ chain V-III region 26370404 .53
Complement C3 997739375 .98 Ig κ chain V-I region 25247608 .50
Ig α-2 chain C region 859136894 .85 Ig λ-1 chain C region 22965004 .46
Osteopontin 625379556 .62 Ig heavy chain V-III region 22335087 .45
Ig λ-2 chain C region 618988102 .61 Ig γ-1 chain C region 16978721 .34
Lactadherin 614760779 .61 Ig κ chain V-II region 13684364 .27
α-1-antichymotrypsin 495889903 .49 Serum albumin 13040560 .26
Ig μ chain C region 459572098 .45 Proactivator polypeptide 12402391 .25

Ig, immunoglobulin. Ig, immunoglobulin.

8 CHEN ET AL
Correlations between individual proteins from in
vitro–digested samples and inflammation/cytotoxicity
The proteins mucin-5B (R = .997; P = .049) and κ-casein (R = .771; P =
.009) in the in vitro–digested samples were significantly positively cor-
related with LPS-induced IL-8 production in Caco-2BBe cells. The pro-
tein HLA class II histocompatibility antigen DR α chain was significantly
positively correlated with TNF-induced IL-8 production (R = .671; P =
.034).
Correlations between protein combinations from in
vitro–digested samples and inflammation/cytotoxicity
Based on multiple regression models, the combination of tenascin and
complement C3 proteins were positively correlated with IL-8 produc-
tion in Caco-2BBe cells without stimulation (R = .834; P = .016). The
proteins Ig κ V-III region SIE and Ig λ 2 chain C regions were positively
correlated with LPS-induced IL-8 production (R = .779; P = .038). In
unstimulated Caco-2BBe cells, the following protein-combined profiles
were positively correlated with cytotoxicity: polymeric immunoglob-
ulin receptor and Ig J chain (R = .852; P = .011); galectin-3 bind-
ing protein, α-lactalbumin, Ig α-2 chain C region, complement C3, and
tenascin (R = .959; P = .026); Ig J chain, galectin-3 binding protein,
and polymeric immunoglobulin receptor (R = .976; P = .002). The
protein combination of complement C3 and galectin-3–binding pro-
tein was positively correlated with LPS-induced cytotoxicity (R = .797;
P = .029).
Protein abundance patterns among different in
vitro–digested colostrum samples
Using heat maps showing abundance of each protein within each
in vitro–digested colostrum samples, the cell/protein regulation and
immune protein groups were in higher abundance than enzymes and
transport protein groups (Figure 3).
DISCUSSION
Most proteins were significantly (>90%) or completely degraded after
in vitro digestion in the colostrum samples. This finding suggests the
need to shift future research away from simply examining the bioactiv-
ity of proteins present in HM to determining the extent of HM protein
survival across digestion and the bioactivities of digestion-resistant
proteins and their released peptide fragments.
Numerous individual proteins and protein combinations were pos-
itively correlated with cellular inflammation and cytotoxicity. How-
ever, more individual proteins and protein combinations were corre-
lated with cellular outcomes in unstimulated or LPS-stimulated cells
than with TNF-stimulated cells. The stimulation conditions for the
Caco-2BBe cells simulate different intestinal conditions: (1) unstim-
ulated cells can be equivalent to baseline intestines without micro-
biome; (2) LPS-stimulated cells simulate an intestinal environment
experiencing some bacterial load; and (3) TNF-stimulated cells simu-
late an intestinal environment in which an insult has occurred with
tissue damage, causing recruitment of acute cytokines. We observed
more proteins and protein combinations were correlated with cellular
outcomes under unstimulated condition, suggesting these digestion-
resistant proteins stimulate basal cellular inflammation under base-
line intestinal condition. Although this observation seems counterin-
tuitive, previous research has shown that low-grade inflammation is
necessary to promote healthy fetal and infant intestinal growth and
development.15,16 We also observed positive correlations between
digestion-resistant proteins in stimulating inflammation and cell death
under LPS-stimulated conditions (intestinal environment with a bacte-
rial load). Similarly, although the data suggest the digestion-resistant
proteins induced cellular inflammation and cell death, this finding is
still consistent with previous research findings that early-life gut micro-
biome, as well as specifically the polysaccharide on certain microorgan-
isms, stimulate intestinal immune response to a balance between type
1 and 2 helper cell responses and activate T-regulatory cell response
(promoting healthy infant immune development and tolerance).17
Although some digestion-resistant proteins were positively correlated
with cytotoxicity under LPS stimulation, the observed percentage of
cytotoxicity was overall lower in LPS-stimulated cells than in unstim-
ulated cells. In our cell culture experiments, although we were able
to measure and calculate percentage of cytotoxicity under the dif-
ferent treatment and stimulation conditions via LDH measurements,
the measurement does not allow for differentiation between home-
ostatic cellular turnover vs induced cell death. It is possible that the
interactions between some digestion-resistant proteins and LPS pres-
ence induced necessary inflammation to promote homeostatic cellular
turnover, thus improving intestinal cell growth and development.
On the other hand, not many digestion-resistant proteins and pro-
tein combinations were correlated with cellular inflammatory and
cytotoxic outcomes after TNF stimulation. Overall, in comparison with
unstimulated and LPS-stimulated cells, TNF stimulation induced five-
to twofold more IL-8 production, respectively, and about twofold
more percentage of cytotoxicity. This observation implies that once
actual cellular injury has occurred with recruitment of inflamma-
tory cytokines (TNF), the potential intestinal cell growth–promoting
digestion-resistant proteins may not be able to offset an active, robust
inflammatory process.
Although we observed significant correlations between the abun-
dance of some individual proteins and combinations of proteins in
colostrum samples on cellular outcomes, we did not examine the direct
effects of individual proteins or protein combinations on cellular out-
comes. Future studies need to investigate the direct effects of these
correlated single and combination proteins on intestinal epithelial cells
to determine if these proteins contribute towards the variations in
cellular protection among colostrum samples from different moth-
ers. In addition, future cell culture–based studies measuring biochem-
ical markers to differentiate between apoptosis vs necroptosis would
be helpful to determine if our suspicion that the observed positive
JOURNAL OF PARENTERAL AND ENTERAL NUTRITION 9

F I G U R E 3 Heat maps showing protein-assigned ion abundance patterns among different in vitro–digested colostrum samples. Proteins were
grouped by bioactivities (indicated by class on the right side and represented on the top of the heat maps): cell/protein regulation (red), enzymes
(green), immune proteins (royal blue), and transport proteins (sea foam blue). Within the heat maps, protein-assigned ion abundance is indicated by
shades of blue and maroon: dark blue is minimal abundance and dark maroon is high abundance. (a) All individual proteins identified (x-axis) within
each colostrum sample (y-axis). Cell/protein regulation and immune proteins are more abundant in digested colostrum samples. (b) Abundance of
each protein group among digested colostrum samples. Similar to Figure 3A, protein groups cell/protein regulation and immune proteins exhibit
higher abundance

correlations between individual proteins and protein combinations and
inflammation/cytotoxicity in unstimulated and LPS-stimulated cells
promoted homeostatic cell turnover (apoptosis) rather than patho-
logic induced cell death (necroptosis), whereas the cellular inflam-
mation and cytotoxicity observed in TNF-induced cells were patho-
logic. This determination can be accomplished by measuring additional
cell death markers, such as terminal deoxynucleotidyl-transferase-
mediated deoxyuridine triphosphate nick-end labeling assay, activated
caspase 3 (apoptosis), and activated RIPK1 and RIPK3 (necroptosis).18
The rationale for examining the effects of individual and combina-
tion proteins on types of cell death is based on two key factors: (1)
the preterm neonatal gut grows and develops at a higher rate com-
pared with the term neonatal gut, likely through increased homeo-
static cell turnover;15 and (2) necroptosis-type cell death with eleva-
tion in intestinal TNF concentrations have been implicated in human
NEC cases.19 Furthermore, it will be ideal to also include concurrent
assessment of cell proliferation, such as 3H-thymidine or 5-bromo-2’-
deoxyuridine labeling, to comprehensively assess the effects of specific
proteins on the overall cell proliferation and cell death types. This com-
bined assessment of cell turnover as a function of various HM proteins
may provide insight into how different women’s HM components are
more or less protective against pathogenic cytotoxicity by shifting the
balance between apoptosis vs necroptosis while optimizing cell prolif-
eration to reduce risk for NEC.
Based on results from this current study, there are likely numer-
ous digestion-resistant proteins that modified cytotoxicity. Specifically,
we observed a larger number of immune and cell/protein regulation
proteins that are resistant to in vitro digestion that may have affected
cytotoxicity. One protein, mucin-5B, was not identified in undigested
colostrum samples but consistently identified in all digested colostrum
samples, and the protein-assigned ion abundance for one other pro-
tein (HLA class II histocompatibility antigen DR α chain) was higher
in 50% of the digested colostrum samples compared with undigested
counterparts. The protein sequence for mucin-5B was checked against
porcine and human-protein databases to confirm the origin of this iden-
tified protein in digested colostrum samples because porcine-derived
digestive enzymes were used for the in vitro–digestion protocol. We
confirmed that the protein sequence was of human origin. A possi-
ble explanation for observing mucin-5B in our digested but not undi-
gested colostrum samples, may be the overall relative abundance of
proteins in undigested samples were at a much higher order of mag-
nitude than digested samples; thus, proteins that are present at much
lower relative abundance may not have been quantified and identified.
The presence and activities of proteins found in the in vitro–digested
samples deserve further investigation because mucin-5B was found to
be positively correlated with IL-8 production by intestinal epithelial
cells induced with LPS, and HLA class II histocompatibility antigen DR
α chain was positively correlated with TNF-induced IL-8 production.
Future studies should examine whether the cells are similarly modu-
lated by purified individual proteins and protein combinations found
to be significantly correlated with cellular outcomes to confirm bioac-
tivity, coupled with the previously mentioned differential cytotoxicity
markers. Additionally, future studies may consider improving the pro-
teomic analysis through increasing sample size, further fractionating
the milk samples, and optimizing LC with tandem MS method to help
identify more proteins in both undigested and digested samples, thus
improving the assessment of protein losses from digestion.
Alhough Caco-2 BBe is a commonly used cell line as a model for
the distal small intestine, it is important to note that the Caco-2 BBe
cell line derives from human colonic adenocarcinoma, thus potentially
limiting the translatability of our results. However, the Caco-2 BBe
cell line continues to be a common cell line model because of its well-
described small intestinal morphological and functional characteristics
when full cell differentiation is achieved.20,21 Nonetheless, even after
10
full differentiation to closely simulate small intestinal enterocytes, this
single-cell type model is still limited in its translatability to the com-
plex multicell type human intestinal environment. Future studies using
the enteroid model may further extend mechanistic investigation and
increase translatability of findings.
Our research group, along with many other HM research groups,
focus on specific components of HM. Although we focus our investiga-
tions on proteins from the aqueous fraction of HM, we acknowledge
that HM samples also contain an array of other bioactive components
(eg, proteins associated with milk-fat globule membrane and extracel-
lular vesicles, milk lipids, and HM oligosaccharides) that likely function
synergistically to improve infant health. A multipart approach is essen-
tial to define the bioactivities of both isolated milk components and
the synergistic complex of milk components that infants receive across
their digestion.
CONCLUSION
Most proteins in colostrum are significantly or completely degraded
after in vitro digestion. Among digestion-resistant proteins, more were
cell/protein regulation and immune proteins than enzymes and trans-
port proteins. More individual proteins and protein combinations were
correlated with unstimulated and LPS-stimulated intestinal epithelial
cell inflammation and cytoxicity compared with TNF-stimulated cells.
Although the correlations between protein(s) and cellular outcomes
were positive in unstimulated and LPS-stimulated cells, TNF stimula-
tion induced overall more robust cellular inflammation and cytotoxic-
ity. The results suggest that some of the individual proteins and protein
combinations may be inducing low-grade inflammation, which could
promote intestinal growth and development by enhancing homeo-
static cellular turnover under unstimulated and LPS-stimulated condi-
tions. The lack of correlations of protein abundances with TNF-induced
inflammation suggests that this type of inflammation and possible
pathogenic cytotoxicity may be too profound for digestion-resistant
proteins to exert any potential bioactivities. The bioactivities of these
individual and combinations of digestion-resistant proteins need to be
tested and confirmed, along with cell death measurements that differ-
entiate between homeostatic cell turnover (apoptosis) vs pathogenic
cell death (necroptosis) often seen in NEC.
Table S1 is available online at http://pen.sagepub.com.
ACKNOWLEDGMENTS
This work was supported by the International Society for Research
on Human Milk and Lactation–Family Larsson-Rosenquist Foundation
Trainee Expansion Program (Y.C.), the K99/R00 Pathway to Indepen-
dence Career Award, Eunice Kennedy Shriver Institute of Child Health
and Development of the National Institutes of Health (R00HD079561)
(D.C.D.), the US Department of Agriculture National Institute of Food
and Agriculture (2018-67017-27521) (D.C.D.), the Gerber Foundation
(2017-1586) (D.C.D.), and the US Department of Agriculture Multi-
state Workgroup W4002.
CHEN ET AL
CONFLICT OF INTEREST
None declared.
AUTHOR CONTRIBUTIONS
Yimin Chen and David C. Dallas conceptualized the project; Yimin
Chen performed all experiments, and Bum Jin Kim performed all mass
spectromy–related experiments; Yimin Chen and David C. Dallas pro-
vided all essential materials; Yimin Chen and Bum Jin Kim analyzed all
the data, and Yimin Chen conducted all statistical analyses; Yimin Chen,
Bum Jin Kim, and David C. Dallas interpreted the data; Yimin Chen
and Bum Jin Kim wrote the manuscript; Yimin Chen, Bum Jin Kim, and
David C. Dallas were responsible for editing and critically revising the
manuscript for important intellectual content; and Yimin Chen, Bum
Jin Kim, and David C. Dallas provided final approval of the version to
be published. All authors agree to be accountable for all aspects of the
work in ensuring that questions related to the accuracy or integrity of
any part of the work are appropriately investigated and resolved.
ORCID
Yimin Chen PhD, RDN https://orcid.org/0000-0003-2246-7587
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SUPPORTING INFORMATION
Additional supporting information may be found in the online version
of the article at the publisher’s website.
How to cite this article: Chen Y, Kim BJ, Dallas DC.
Proteomics analysis reveals digestion-resistant proteins from
colostrum are associated with inflammatory and cytotoxic
responses in intestinal epithelial cells. Journal of Parenteral and
Enteral Nutrition. 2021;1-11.
https://doi.org/10.1002/jpen.2285