TABLE 2.

Recent studies using proteomic techniques highlighting the beneficial role of carotenoids and their potential impact on relevant health outcomes

Study [reference] and main findings	Model	Technique	Design (approach)		Carotenoid	Dosage (duration)	Tissue	Disease state
In vitro studies
Proteomic response of inflammatory stimulated intestinal epithelial cells to in vitro digested plums and cabbages rich in carotenoids and polyphenols [55]	Caco-2 monolayer; Caco-2/HT-29-MTX (90:10 v/v) with THP-1 macrophages	2D-DIGE	N/A (Untargeted)		Derived from food source	Italian plum (carotenoid content: 1.9 mg/100 g) Kale (carotenoid content: 13.3 mg/100 g) (time of cellular exposure-18h)	Model of intestine	Model of IBD
Findings	Monoculture: 68 protein-spots were regulated due to the Kale and Italian plum digesta Coculture: 206 protein-spots were regulated due to the Kale and Italian plum digesta Some of the identified proteins included antioxidant enzymes such as catalase, superoxide dismutase and glutathione-S-transferases
Carotenoid exposure of Caco-2 intestinal epithelial cells did not affect selected inflammatory markers but altered their proteomic response [56]	Caco-2	2D-DIGE	N/A (Untargeted)		Lycopene, β-carotene	10 to 25 μg/mL (time of cellular exposure-4h).	Model of intestine	Model of IBD
Findings	β-carotene regulated 15 proteins (involved in antioxidant mechanisms, such as GSTA1) Lycopene regulated 1 protein (profilin-1)
Unraveling the neuroprotective mechanisms of carotenes in differentiated human neural cells: biochemical and proteomic approaches [151]	SH-SY5Y human neuroblastoma cells	LC-MS/MS (Q-TOF LC/MS)	N/A (Targeted)		Total carotene extract (TMC): α-carotene, β-carotene, γ-carotene, lycopene	0.1 μg/mL (24 h)	Bone Marrow	Oxidative stress
Findings	Total mixed carotenoid treatment modulated: RPS: ↓ RPS9, RPS16, RPS4X, RPS19 Tubulins: ↑ TUBA1B, TUBB3, TUBB4B, TUBB6, TUBB2B PDI: P4HB (PDIA1), PDIA3, PDIA6 HSP: HSPA9 (HSP70), HSPD1 (HSP60) and HSP90AB1 (HSP90β), ↓ ROS, ↑ SOD, ↑ CAT
Systematic investigation of lycopene effects in LNCaP cells by use of novel large-scale proteomic analysis software [152]	LNCaP cells	ICAT ESI-MS/MS and a linear ion trap-FT ion cyclotron resonance mass spectrometer		N/A (Targeted)	Lycopene	0.2 μM (48 h)	Prostate	Chemo-prevention
Findings	Lycopene upregulated a group of enzymes associated with the antioxidant response element, involved in detoxification of ROS, including EPHX1, SOD-1, CAT, and TF
In Vivo Animal Studies
Torularhodin alleviates hepatic dyslipidemia and inflammations in high-fat diet-induced obese mice via PPARα [153]	Male C57BL/6J mice	HPLC/MS (TMT-labeled peptides)	3 groups (n=10/group): 1st group: Control 2nd group: HFD (45% high fat diet) 3rd group: HFD-torularhodin (Targeted)		Torularhodin	40 mg/kg/d in diet (12 wk)	Liver/ adipose tissue	Hepatic dyslipidemia and obesity
Findings	Torularhodin: ↑ anti-inflammation proteins (FAS, BAX, ICAM1, OCLN, GSTP1, FAF1, LRP1, APEX1, ROCK1, MANF, STAT3, and INSR) ↓ proinflammatory proteins (OPTN, PTK2B, FADD, MIF, CASP3, YAP1, DNM1L, and NAMPT)
Proteomic responses of carotenoid and retinol administration to Mongolian gerbils [11]	Gerbils (n=30)	2D-DIGE LC-MS/MS	1st group (n=6) all-trans β-carotene 2nd group (n=6) lycopene 3rd group (n=6) lutein 4th group (n=6) retinol 5th group (n=6) vehicle (control)(Untargeted)		β-Carotene, lutein, lycopene, retinol	Single dose of 100 mg/kg bw (retinol: 53 mg/kg bw) given by oral gavage (12 h)	Liver, white adipose tissue (WAT), plasma	Proteomic response
Findings	Carotenoids regulated: 29 protein types in the liver, in the field of protein metabolism, lipid metabolism, detoxification, transport, and energy metabolism. 19 protein types in WAT, involved in cell structure, energy metabolism and lipid metabolism. 66 protein types in plasma, involved in the immune system/inflammation and protein metabolism. Carotenoid administration affected cell structure (22), protein metabolism (15), and immune system/inflammation (19) Similar proteins differentially regulated by lycopene compared to retinol
In vivo human studies
Plasma Proteins associated with circulating carotenoids in Nepalese school-aged children [10]	6-8 y old Nepalese children	iTRAQ	500 samples randomly chosen from a 5-arm antenatal micronutrient supplementation trial of 6–8 y old Nepalese children [247] (Untargeted)		β-Carotene, lutein and zeaxanthin, β-cryptoxanthin, α-carotene, and lycopene	Samples from a 5-arm antenatal micronutrient supplementation trial [247]	Plasma	Vitamin A deficiency
Findings	4 plasma proteins were associated with β-carotene 11 plasma proteins were associated with lutein/zeaxanthin 51 plasma proteins were associated with β-cryptoxanthin No protein biomarkers associated with α-carotene or lycopene Plasma proteins were notably involved in lipid and vitamin A transport, antioxidant function and anti-inflammatory processes
Plasma proteome fingerprint associated with circulating carotenoids and retinol in older adults [54]	728 adults (≥65)	SomaScan assay	Cross-sectional analysis (Untargeted)		α-Carotene, β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene	Samples from a population-based study [248]	Plasma	Aging
Findings	Plasma proteomic fingerprint associated with elevated circulating carotenoids in older adults were related to: Sirtuin signaling (NAMPT) Inflammation and oxidative stress (CCNB1, SOD2, IL1RAP, CNDP1) Iron metabolism (HAMP, ferritin) Proteostasis (CLU, CTSV, ACY1) Innate immunity (FCN2) Longevity (CRP, GDF15, THBS2)

Abbreviations: 2D-DIGE, 2-dimensional difference gel electrophoresis; ACY1, aminoacylase 1; APEX1, apurinic/apyrimidinic endonuclease 1; BAX, Bcl-2–associated X; CASP3, cysteine-aspartic acid protease 3; CAT, catalase; CCNB1, cyclin B1; CLU, clusterin; CNDP1, carnosine dipeptidase 1; CRP, C-reactive protein; CTSV, cathepsin V; DNM1L, dynamin-1-like protein; EPHX1, epoxide hydrolase 1; FADD, Fas-associated death domain; FAF1, Fas associated factor 1; FCN2, ficolin-2; GDF15, growth/differentiation factor 15; GST, glutathione S-transferase; HAMP, hepcidin antimicrobial peptide; HFD, high fat diet; HSP, heat shock protein; IBD, inflammatory bowel disease; ICMA1, intercellular adhesion molecule 1; IL1RAP, interleukin-1 receptor accessory protein; INSR, insulin receptor; LC, liquid chromatography; LRP1, LDL receptor related protein 1; MANF, mesencephalic astrocyte-derived neurotrophic factor; MIF, macrophage migration inhibitory factor; MS, mass spectrometry; NAMPT, nicotinamide phosphoribosyltransferase; OCLN, occludin; PDI, protein disulfide isomerases; PTK2B, protein tyrosine kinase 2-beta; Q-TOF, quadrupole-time of flight; ROCK1, Rho-associated protein kinase 1; RPS, ribosomal protein; SOD, superoxide dismutase; TF, transferrin; THBS2, thrombospondin-2; YAP1, Yes-associated protein 1.