Proteomic characterization of the acid-insoluble fraction of whole saliva from preterm human newborns.
The acid-insoluble salivary proteome obtained by addition of TFA to whole human saliva from adults, preterm and at-term newborns has been analysed by 2-DE in order to evidence differences among the three groups, and integrate data previously obtained on the acid-soluble fraction. 2-DE spots differentially expressed among the three groups were submitted to in-gel tryptic digestion and the peptide mixtures analysed by high resolution HPLC–ESI–MS/MS. By this strategy, we identified 3 over-expressed proteins in at-term newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100 A6, S100 A7, S100 A9, prolactin-inducible protein, Ig kappa chain, cystatin SN, cystatin S/SA and α-amylase 1). Four spots, already detected but not characterized by other authors in human saliva 2-DE, were attributed to different protein species of S100 A9 (long-type and long-type monophosphorylated, short-type and short-type monophosphorylated) by MS/MS analysis of tryptic peptides and sequential staining of 2-DE gels with Pro-Q Diamond, for specific detection of phosphoproteins, and total protein SYPRO Ruby stain.



Differential protein expression analysis of the acid insoluble fraction of saliva from preterm, at-term newborns and adults has been performed in this study by coupling 2-DE analysis and high-resolution tandem mass spectrometry in order to complete the information previously obtained by top-down LC–MS only on the acid-soluble proteome. Several proteins identified in the acid insoluble fraction of both preterm newborn and adult saliva are not of glandular origin, being only prolactin-inducible protein, salivary cystatins, α-amylase and polymeric immunoglobulin receptor exclusive of salivary glands. 

Three proteins resulted increased in at-term newborns with respect to preterm newborns and adults: BPI fold-containing family A member 1, two proteoforms of annexin A1 and keratin type 1 cytoskeletal 13, while several proteins were significantly increased in adults.
Department of Life and Environmental Sciences, Biomedical Section, University of Cagliari, Monserrato Campus, 09042 Monserrato, CA, Italy.
Morena
M
Arba
Biochemistry and Clinical Biochemistry Institute, Catholic University of Rome, L.go F. Vito, 1, 00168 Rome, Italy.
Federica
F
Iavarone
Biochemistry and Clinical Biochemistry Institute, Catholic University of Rome, L.go F. Vito, 1, 00168 Rome, Italy.
Federica
F
Vincenzoni
Department of Life and Environmental Sciences, Biomedical Section, University of Cagliari, Monserrato Campus, 09042 Monserrato, CA, Italy.
Barbara
B
Manconi
Institute of Clinical Pediatrics, Catholic University of Rome, L.go F. Vito,1, 00168 Rome, Italy.
Giovanni
G
Vento
Institute of Clinical Pediatrics, Catholic University of Rome, L.go F. Vito,1, 00168 Rome, Italy.
Chiara
C
Tirone
Department of Life and Environmental Sciences, Biomedical Section, University of Cagliari, Monserrato Campus, 09042 Monserrato, CA, Italy.
Tiziana
T
Cabras
Biochemistry and Clinical Biochemistry Institute, Catholic University of Rome, L.go F. Vito, 1, 00168 Rome, Italy; Institute of Chemistry of the Molecular Recognition, CNR, L.go F. Vito, 1, 00168 Rome, Italy.
Massimo
M
Castagnola
Institute of Chemistry of the Molecular Recognition, CNR, L.go F. Vito, 1, 00168 Rome, Italy.
Irene
I
Messana
Department of Life and Environmental Sciences, Biomedical Section, University of Cagliari, Monserrato Campus, 09042 Monserrato, CA, Italy. Electronic address: sanna@unica.it.
Maria Teresa
MT
Sanna
D000293:Adolescent; D000328:Adult; D017305:Annexin A1 / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007234:Infant, Premature / Q000378:metabolism*; D053556:Keratin-1 / Q000032:analysis; D008875:Middle Aged; D010750:Phosphoproteins / Q000032:analysis; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D012463:Saliva / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D055815:Young Adult
D017305:Annexin A1; C485599:BPIFA1 protein, human; D006023:Glycoproteins; C491616:KRT1 protein, human; D053556:Keratin-1; D010750:Phosphoproteins; D020543:Proteome
2-dimensional electrophoresis; Human saliva; Mass spectrometry; Preterm newborn; Tandem mass spectrometry