PMID- 20187109
OWN - NLM
STAT- MEDLINE
DCOM- 20100629
LR  - 20100325
IS  - 1552-4930 (Electronic)
IS  - 1552-4922 (Linking)
VI  - 77
IP  - 4
DP  - 2010 Apr
TI  - Single amino acid resolution of proteolytic fragments generated in individual 
      cells.
PG  - 347-55
LID - 10.1002/cyto.a.20880 [doi]
AB  - The complex nature of enzyme regulation mandates that enzyme activity profiles be 
      measured in the context of the intact cell. Single-cell capillary electrophoresis 
      (CE) coupled with laser-induced fluorescence is a powerful approach for 
      quantitation and separation of analytes present in small samples and single live 
      cells; however, it does not allow for the definitive identification of the 
      reaction products. On the other hand, mass spectrometry (MS) is able to identify 
      analytes but still lacks the requisite sensitivity for most single-cell analysis 
      applications. Thus, it follows that by determining the relative amounts of 
      reaction products generated in single cells using CE and by producing larger 
      quantities of these products using bulk cell populations to identify them using 
      MS, it is possible to determine enzyme activity profiles in single cells. In this 
      study, the applicability of this approach was demonstrated by examining the 
      intracellular fate of a protease substrate derived from the beta-amyloid 
      precursor protein (beta-APP). In single live TF-1 cells, three distinct fragments 
      were generated from the beta-APP peptide, which differed by a single uncharged 
      amino acid. The CE measurements indicated that the proteolytic fragment profiles 
      (i.e., the relative amounts of each fragment) were consistent from cell to cell 
      but that they were different from those obtained in cell lysates. Furthermore, 
      measurements obtained at the single cell level made it possible to observe a 
      modest but statistically significant negative correlation between the total 
      amount of beta-APP peptide loaded in cells and the fraction of peptide that 
      remained intact. This study demonstrates how single-cell CE, MS, and peptide 
      substrates can be combined to identify and measure enzyme activities in single 
      live cells.
FAU - Brown, Robert B
AU  - Brown RB
AD  - Institute of Biomaterials and Biomedical Engineering (IBBME), University of 
      Toronto, Toronto, Ontario, Canada.
FAU - Hewel, Johannes A
AU  - Hewel JA
FAU - Emili, Andrew
AU  - Emili A
FAU - Audet, Julie
AU  - Audet J
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Cytometry A
JT  - Cytometry. Part A : the journal of the International Society for Analytical 
      Cytology
JID - 101235694
RN  - 0 (Amino Acids)
RN  - 0 (Amyloid beta-Protein Precursor)
RN  - 0 (Cell Extracts)
RN  - 0 (Peptide Fragments)
SB  - IM
MH  - Amino Acid Sequence
MH  - Amino Acids/*analysis
MH  - Amyloid beta-Protein Precursor/metabolism
MH  - Cell Extracts
MH  - Cell Line, Tumor
MH  - Cell Survival
MH  - Cells/*metabolism
MH  - Chromatography, Liquid
MH  - Electrophoresis, Capillary
MH  - Humans
MH  - Mass Spectrometry
MH  - Molecular Sequence Data
MH  - Peptide Fragments/*analysis/chemistry/isolation & purification
MH  - *Protein Processing, Post-Translational
EDAT- 2010/02/27 06:00
MHDA- 2010/06/30 06:00
CRDT- 2010/02/27 06:00
PHST- 2010/02/27 06:00 [entrez]
PHST- 2010/02/27 06:00 [pubmed]
PHST- 2010/06/30 06:00 [medline]
AID - 10.1002/cyto.a.20880 [doi]
PST - ppublish
SO  - Cytometry A. 2010 Apr;77(4):347-55. doi: 10.1002/cyto.a.20880.