Methods

GeLC/MS is a powerful but simple approach for proteomic analyses. Samples are separated using 1-D SDS-PAGE. Post separation the gel lane is divided into equally sized segments. Proteins in each segment are reduced and alkylated, and in-gel digestion performed using a site-specific protease such as trypsin. An LC-MS/MS experiment is then performed to obtain peptide sequence information and hence identify the proteins in each segment. Data are collated in a non-redundant list per gel lane.

The steps involved in a GeLC/MS experiment are:

• Sample preparation
• SDS-PAGE
• Segmentation
• Automated in-gel digestion
• NanoLC/MS/MS
• Data analysis - including Mascot, SEQUEST, and Scaffold

Ref: "Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry." M. Wilm, A. Shevchenko, T. Houthaeve, S. Breit, L. Schweigerer, T. Fotsis, M. Mann. Nature 1996, 379, 466-469.

Ref: "Profiling Core Proteomes of Human Cell Lines by One-dimensional PAGE and Liquid Chromatography-Tandem Mass Spectrometry" M. Schirle, M. Heurtier, B. Kuster. Molecular and Cellular Proteomics 2003, 2, 1297-1305

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Multiple Reaction Monitoring (MRM) is considered the most selective and sensitive mass spectrometry scan function. The application of LC/MRM-MS as a tool for the quantitation of proteins in complex biological matrices such as plasma, urine and CSF is an active area of research and development. Nextgen Sciences has been successfully applying LC/MRM-MS to the analysis of endogenous protein biomarkers and accordingly optimizing their workflows for work of this nature.

LC-MRM/MS protein assays involve enzymatic digestion of proteins to peptides amenable to MRM analysis. Peptides that act as surrogate or signature of the intact protein are judiciously selected. Nextgen Sciences protein assay development workflow uses fragmentation data derived from LC/MS experiments mapping experiments of a broad range of biological samples. Data are stored in an internal database (biomarkerlibrary™). The database can be referenced with a bioinformatics tool that performs matrix and background selectivity tests and produces candidate peptide lists for a quantitative assay.

Assays are designed to be suitable for all stages of biomarker investigations and assay validation is performed to the extent required for the purpose of the assay.

The steps involved in developing a MRM protein assay are:

• Selection of surrogate or signature peptides
• Sample preparation
• Digestion of proteins
• Iterative testing of peptide and transitions by LC-MRM/MS
• Assay validation
• Assay testing SOP/Method documentation

The steps involved in using a MRM assay to monitor the levels of proteins are:

• Sample preparation
• Digestion of proteins
• LC-MRM/MS
• Data analysis
• Report preparation

Ref: "Absolute Quantification of the Model Biomarker Prostate-Specific Antigen in Serum by LC−MS/MS Using Protein Cleavage and Isotope Dilution Mass Spectrometry". D.R. Barnidge, M.K. Goodmanson, G. G. Klee, and D.C. Muddiman. Journal of Proteome Research, 2004, 3, 644-652.

Ref: "Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins". L. Anderson and C. Hunter, Molecular and Cellular Proteomics, 2006, 5, 573-88

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Differential mass spectrometry (dMS) is a general proteomics workflow that provides relative quantation and identifies statistically significant changes in full scan mass spectrometry data. dMS is a quantitative proteomics approach that requires no metabolic or chemical labelling, or sample mixing. As such dMS data from one sample can be compared to data from many others. The absence of mixing or labelling make dMS ideally suited to the analysis of complex samples such as tissue homogenates or body fluids.

In a dMS experiment LC/MS data are acquired using the full scan mode of operation. Post acquisition data processing is used to collate a ranked list of differences between samples in multiple conditions. Experiments are designed to account for analytical, technical and biological variation among data points and samples.

The steps involved in a dMS experiment are:

• Sample preparation. In-solution digests are prepared of samples that may or may not have undergone some form of depletion of abundant proteins.
• LC/MS. Optimization of chromatographic conditions enables dMS without system related bias
• Data Analysis. Performed using the Thermo SEIVE™ software

Ref: "Differential Mass Spectrometry: A Label-Free LC-MS Method for Finding Significant Differences in Complex Peptide and Protein Mixtures" M. C. Wiener, J.R. Sachs, E.G. Deyanova. N.A. Yates. Analytical Chemistry, 2004, 76, 6085-6096

Ref: "Differential Mass Spectrometry of Rat Plasma Reveals Proteins that are Responsive to 17-Estradiol and Selective Estrogen Receptor Modulator PPT" X. Zhao, E.G. Deyanova, L.S. Lubbers, P.Zafian, J.J. Li, A. Liaw, Q. Song, Y. Du, R.E. Settlage, G.J. Hickey, N.A. Yates, R.C. Hendrickson. Journal of Proteome Research 2008, 7, 4373-4383

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Stable isotope labeling by amino-acids in cell culture (SILAC) is a stable isotope labeling strategy. Labeled essential amino acids are added to amino acid deficient cell culture media which are therefore incorporated into all proteins as they are synthesized "encoded into the proteome". No chemical labeling or affinity purification steps are performed, and the method is compatible with virtually all cell culture conditions, including primary cells.

The steps involved in a SILAC experiment are:

• Sample preparation
• SDS-PAGE
• Segmentation
• In-gel digestion
• Nano LC-MS/MS
• Data analysis

Ref: "Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics". S. Ong, B. Blagov, I. Kratchmarova, D.B. Kristensen, H. Steen, A. Pandey, M. Mann. Molecular and Cellular Proteomics 2002, 1, 376-386.

Ref: "Quantitative Cancer Proteomics: Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) as a Tool for Prostate Cancer Research". P. A. Everley, J. Krijgsveld, B.R. Zetter, S.P Gygi, S. P. Molecular and Cellular Proteomics 2004, 3, 729-735.

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Difference gel electrophoresis (DIGE) utilizes mass- and charge-matched, spectrally resolvable fluorescent dyes (Cy3 and Cy5) to label two different protein samples in vitro prior to 2-D gel electrophoresis. Compared to conventional 2D gel electrophoresis, DIGE has the major advantage that both the control and experimental sample are run in the same gel. These samples are then imaged separately but because they were run in the same gel, the images can be perfectly overlaid without "warping". This reduces the number of gels that require "warping" by imaging software.

The steps involved in a DIGE experiment are:

• Sample preparation including labeling of proteins with Cy dyes
• 2 dimensional gel electrophoresis
• Image analysis
• Spot excision
• In-gel protein digestion
• LC-MS/MS

Ref: "Difference gel electrophoresis: a single gel method for detecting changes in protein extracts". M. Unlu, M.E. Morgan, J.S. Minden. Electrophoresis 1997, 18, 2071-2077.

Ref: "Multivariable Difference Gel Electrophoresis and Mass Spectrometry: : a case study on transforming growth factor-beta and ERBB2 signaling". D.B. Friedman, S.E. Wang, C.W. Whitwell, R.M. Caprioli, C.L. Arteaga. Molecular and Cellular Proteomics 2007, 6, 150-169.

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Sodium dodecylsulfate polyacrylamide gel electrophoresis is the most commonly used low level protein separation and purification technique in biological research. The reason for using this technique in LC/MS is to reduce sample complexity and to purify sample contaminants as detergents, salts, DNA and other protein material.

There are several options for 1-D gel electrophoresis/SDS-PAGE include:

• Mini and large formats
• Varying composition of gel suited to different separations

Ref: "Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels". A.L. Shapiro, E. Vinuela, J.V. Maizel. Biochemical and Biophysical Research Communications 1967, 28, 815-820.

Ref: "The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis." K. Weber, M. Osborn. Journal of Biological Chemistry. 1969, 244 , 406-4412.

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