Table II.

Candidate MMP-9 substrates identified by subtractive proteomics in macrophage-conditioned medium

Macrophage proteins were identified by MS/MS analysis as described in the legend to Table I. Candidate MMP-9 substrates were identified by requiring (i) at least 3 times as many unique peptides in medium from M9A macrophages as in medium from WT macrophages, (ii) detection of at least two unique peptides in two of three independent analyses, (iii) a medium/lysate ratio of >1.5, and (iv) a significant difference in the number of unique peptides between M9A cells and WT cells as assessed by Student's t test or Bolshev's sign test. Results represent means of three independent experiments. Y, yes; N, no.

Entry name	Protein name	Average no. of unique peptides		Structural annotation		Statistical significance
		M9A	WT	Signal peptidea	TM domaina	Bolshev's test	t test
Membrane-associated proteins
GLG1_MOUSE	Golgi apparatus protein 1 precursor (E-selectin ligand 1) (ESL-1)	2.7	0.0	Y	Y	+b	0.06
A4_MOUSE	Amyloid-β A4 protein precursor (APP)	11.3	2.3	Y	Y	+	0.005
ITGB2_MOUSE	Integrin β2 precursor (CD18)	4.0	1.0	Y	Y	+	0.02
IGSF8_MOUSE	Immunoglobulin superfamily member 8, CD316 antigen	4.0	1.0	Y	Y	+	0.02
RAE1A_MOUSEc	Retinoic acid early inducible protein 1 α precursor (RAE-1α)	3.0	0.0	Y	N	+	0.19
Secreted proteins
HEXB_MOUSE	β-Hexosaminidase β chain precursor	6.7	0.0	Y	N	+	0.11
RISC_MOUSE	Retinoid-inducible serine carboxypeptidase precursor	3.3	0.0	Y	N	+	0.13
Q61297_MOUSE	Pancreatic α-amylase isozyme	3.0	0.0	Y	N	+	d
ARSB_MOUSE	Arylsulfatase B precursor	2.7	0.0	Y	N	+	0.18
MA2B2_MOUSE	Epididymis-specific α-mannosidase precursor	2.0	0.0	Y	N	+	0.23
MMP9_MOUSE	Matrix metalloproteinase-9 (MMP-9, gelatinase B)	70.0	2.0	Y	N	+	0.00002
Q8BK91_MOUSE	N-Acetylglucosamine-6-sulfatase precursor	5.7	0.7	?	N	+	0.006
CATE_MOUSE	Cathepsin E precursor	1.7	0.3	Y	N	−b	0.047
AMYS_MOUSE	α-Amylase, salivary and hepatic precursor	27.7	6.3	Y	N	+	0.06
O88325_MOUSE	α-N-Acetylglucosaminidase	4.3	1.0	Ye	?	+	0.38
LICH_MOUSE	Lipase A (cholesteryl esterase)	6.7	1.7	Y	N	+	0.31
BGLR_MOUSE	β-Glucuronidase precursor	14.7	4.7	Y	N	+	0.21
PRDX4_MOUSE	Peroxiredoxin 4 (Prx-IV)	8.7	2.0	Yf	N	+	0.079
PLF4_MOUSE	Platelet factor 4 precursor (PF-4) (CXCL4)	2.0	0.7	Y	N	−	0.057
Proteins lacking a signal peptide or membrane-spanning domain
RL6_MOUSE	60 S ribosomal protein L6	2.7	0.7	N	N	−	0.013

^aAnnotation based on UniProt Knowledgebase.

^b+ or −, passes or fails (Bolshev's sign test at α = 0.05), respectively.

^cGlycosylphosphatidylinositol-membrane anchored.

^dNo peptide identified in WT cells.

^eAnnotation based on Gene Ontology.

^fComputational prediction (Phobius and Sig-Pred).