Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils.
BACKGROUND
Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods.


RESULTS
To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis.


CONCLUSIONS
Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.
Jethwaney
Deepa
D
Buck Institute for Age Research, Novato, CA 94945, USA. bgibson@buckinstitute.org.
Islam
Md Rafiqul
MR
Leidal
Kevin G
KG
de Bernabe
Daniel Beltran-Valero
DB
Campbell
Kevin P
KP
Nauseef
William M
WM
Gibson
Bradford W
BW
J Cell Biol. 1983 Jul;97(1):52-61
6408102
J Leukoc Biol. 2002 Jun;71(6):973-80
12050182
J Leukoc Biol. 1994 Jul;56(1):80-7
7517990
J Immunol. 1994 Jul 15;153(2):804-10
8021514
Blood. 1994 Mar 15;83(6):1640-9
8123855
Biochem J. 1994 Apr 15;299 ( Pt 2):473-9
8172608
Eur J Haematol. 1993 Oct;51(4):187-98
8243606
J Exp Med. 1993 Dec 1;178(6):1935-46
8245774
Eur J Biochem. 1993 Jul 1;215(1):105-11
8344271
J Clin Invest. 1993 Sep;92(3):1467-76
8376598
Int J Biochem Cell Biol. 1997 Feb;29(2):271-4
9147127
J Biol Chem. 1997 May 23;272(21):13793-802
9153235
Blood. 1997 May 15;89(10):3503-21
9160655
Clin Infect Dis. 1997 Sep;25(3):551-73
9314442
Histochem Cell Biol. 1997 Sep;108(3):221-33
9342616
J Clin Invest. 1998 Jan 15;101(2):455-63
9435318
Proteomics. 2002 Jan;2(1):36-47
11788990
Biochem Pharmacol. 1999 Jun 1;57(11):1209-14
10230763
Nat Biotechnol. 1999 Jul;17(7):676-82
10404161
Neurology. 1999 Sep 22;53(5):1119-22
10496277
Electrophoresis. 1999 Dec;20(18):3551-67
10612281
J Immunol Methods. 1999 Dec 17;232(1-2):131-43
10618515
J Immunol Methods. 1999 Dec 17;232(1-2):145-52
10618516
J Leukoc Biol. 2000 Dec;68(6):785-92
11129645
Immunol Rev. 2000 Oct;177:195-203
11138776
J Cell Biol. 2001 Jan 8;152(1):165-80
11149929
Neuromuscul Disord. 2001 Jan;11(1):20-6
11166162
J Leukoc Biol. 2001 Jan;69(1):57-62
11200068
J Exp Med. 2001 Apr 16;193(8):955-66
11304556
J Leukoc Biol. 2001 Oct;70(4):669-76
11590205
Histochem Cell Biol. 2002 Aug;118(2):117-25
12189515
J Biol Chem. 2002 Nov 8;277(45):43399-409
12202484
Nat Rev Immunol. 2003 Feb;3(2):169-76
12563300
Acta Neuropathol. 2003 Jun;105(6):537-42
12734659
Nature. 2003 May 8;423(6936):168-72
12736685
Microbes Infect. 2003 Nov;5(14):1317-27
14613775
Trends Cell Biol. 2004 Apr;14(4):206-13
15066638
Anal Chem. 2004 Jul 15;76(14):4193-201
15253663
J Biol Chem. 2004 Sep 17;279(38):39989-98
15262974
Histochem Cell Biol. 2004 Oct;122(4):277-91
15293055
Blood. 2004 Dec 15;104(13):4269-78
15328157
Nat Rev Microbiol. 2004 Sep;2(9):727-38
15372083
Mol Pharmacol. 1992 Feb;41(2):267-72
1538707
Mol Cell Proteomics. 2005 Jan;4(1):84-96
15591592
J Leukoc Biol. 2005 May;77(5):598-625
15689384
J Leukoc Biol. 2005 Jun;77(6):1008-17
15758076
Mol Cell Proteomics. 2005 Oct;4(10):1503-21
15985654
Cell Mol Life Sci. 2005 Oct;62(19-20):2228-40
16091845
Biochem J. 2006 Jan 1;393(Pt 1):211-8
16144515
J Biol Chem. 2006 Jan 6;281(1):129-36
16275640
J Biol Chem. 1992 Jul 25;267(21):14912-7
1634531
J Leukoc Biol. 2006 Mar;79(3):564-73
16387844
Mol Cell Proteomics. 2006 Apr;5(4):620-34
16415295
Nat Rev Immunol. 2006 Mar;6(3):173-82
16498448
J Biol Chem. 1990 Jan 25;265(3):1436-42
2104841
Nature. 1990 Jan 18;343(6255):282-4
2300173
Blood. 1985 Apr;65(4):964-73
2579692
Proc Natl Acad Sci U S A. 1987 Nov;84(21):7393-7
3118366
Blood. 1983 Mar;61(3):483-92
6297637
J Cell Sci. 2002 Mar 15;115(Pt 6):1321-30
11884531
Nature. 2002 May 2;417(6884):91-4
12018205
J Biol Chem. 1984 Jan 10;259(1):47-52
6706948