Services

LIQUID CHROMATOGRAPHY AND TANDEM MASS SPECTROMETRY ( LC-MS/MS )

Biostring Inc. offers mass spectrometry services for protein identification, protein quantitation and post translational modification (PTM) mapping. Biostring Inc. utilizes high performance nano liquid chromatography (LC) and Tandem mass spectrometry (MS/MS) technologies to achieve unsurpassed resolution and identification accuracy. We can tailor the LC phase to analyze proteins from a simple gel plug to a complex sample like cell lysate or plasma. To increase coverage of protein identification when dealing with a complex sample like serum, plasma, whole cell lysate and tissue homogenate we utilize Multi-Dimensional Protein Identification Technology (MudPIT) to reduce sample complexity prior to LC-MS/MS.

Quantitative protein analysis is another significant area offered along with simultaneous protein identification. We can do both the label-free approach (spectral counting or ion intensity measurements) and the labeled approach using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and Tandem Mass Tags (TMT). We have successfully finished multiple projects utilizing either the label-free approach or iTRAQ and TMT based biomarker identifications and we provide results either as relative abundance (iTRAQ or TMT ratios) or absolute quantities (pmol, pg, ug or fg).

Comprehensive identification of post translational modifications (PTM) is the third most sought after service at Biostring Inc.. We offer simple PTM analysis utilizing a multistage activation approach. For extensive PTM analysis we use either immobilized metal affinity chromatography (IMAC) or Titanium dioxide (TiO2) for phosphopeptide enrichment. For very complex samples, Biostring Inc. starts with Strong Cation Exchange (SCX) fractionation followed by IMAC or TiO2 phosphoenrichment. For lysine rich proteins or proteins without/minimal trypsin cleavable sites, we utilize a variety of nontraditional enzymes like LysC, ArgC, AspN and GluC for good coverage and PTM results.

To ensure that proteins are sufficiently digested into peptides prior to LC-MS/MS, ITSI-Biosciences utilizes the proprietary ProDM kit to accurately calculate the percentage of proteins digested into peptides.

Our team can perform global proteomics, protein expression profiling, comparative proteomics and targeted proteomics for biomarker discovery and monitoring. Most biological and clinical samples can be analyzed directly or after electrophoresis (e.g. 2D-DIGE, SDS-PAGE) and candidate proteins can be identified by nano LC/MS/MS after in-gel or in-solution digestion.

Our Protein Identification and proteomics services include experimental design, sample preparation, tandem mass spectrometry analysis using nano LC-MS/MS, database search, identified protein annotation and report preparation with suggested next steps.

We offer the following services:

NAME

SERVICE DESCRIPTION

UNIT COST

Sample Preparation 1

Homogenization, protein quantitation using ToPA Bradford protein assay kit.

Per sample

Sample preparation 2

Includes, precipitation, buffer exchange, protein quantitation, etc. and the use of ToPI-DIGE, ToPREP and ToPA.

Per sample

Sample preparation 3

Depletion of abundant protein e.g. bovine or human serum albumin using ITSI ASK-Column kit.

Per sample

Sample preparation 4

Depletion of abundant protein e.g. bovine or human serum albumin using ITSI ASK-Solvent kit.

Per sample

Sample preparation 5

Depletion IgY Column Depletion of 14 abundant proteins from serum / plasma using IgY column.

Per sample

Sample preparation 6

Phospho-protein/ peptide enrichment using TiO2 or IMAC technology.

Per sample

Sample preparation 7

In-gel digestion. Digestion of proteins in a gel band/spot using trypsin. The service includes a) gel de-staining – coomassie or silver stain b) alkylation and reduction    c) trypsin digestion d) peptide extraction from the gels for mass spectrometry analysis.

Per sample

Sample preparation 8

In-solution digestion. Digestion of proteins in solution using trypsin. The service includes; a) determining protein concentration (if possible), b) alkylation and reduction c) digestion d) peptide extraction for mass spectrometry analysis.

Per sample

Sample preparation 9

Urine protein isolation and concentration. Service includes Isolation, partial purification, and concentration of urine proteins using ITSI Urine Protein Isolation kit.

Per sample

Sample preparation 10

Zip tip cleanup of in-gel or in-solution digested samples….

Per sample

Sample preparation 11

Any other sample preparation.

Per sample

Flash Proteomics Service (FPS) by tandem mass spectrometry

The Flash Proteomics Service involves the use of validated pre-fractionation protocols and high performance tandem mass spectrometers to rapidly generate a global qualitative protein signature of a moderately-complex specimen. With FPS the run time is 30 minutes or less and most results will be ready in about 48h.Samples including fractions from HPLC, pre-fractionated samples such as protein lysates, serum, plasma and culture media can be screened. In many cases,results will be    ready in 48 hours. This service is ideal for preliminary data generation, to support grant applications, biomarker research, search for contaminants in samples, verification of the integrity and purity of samples, characterization of antibodies and recombinant proteins.

The standard FPS service includes:

  • Sample preparation,
  • Nano-LC/MS/MS.
  • Database search and identified protein annotation.

Per sample

LC60-MS/MS

Peptide sequencing and protein identification by liquid chromatography – tandem mass spectrometry of pure protein samples. Services include

  • Tandem mass spectrometry analysis using nano-LC/MS/MS,
  • Database search and
  • Identified protein annotation – 60 min run.

Per sample

LC90-MS/MS

Peptide sequencing and protein identification by liquid chromatography – tandem mass spectrometry of slightly complex samples, including proteins isolated from gel plugs and gel bands. Services include

  • Tandem mass spectrometry analysis using nano-LC/MS/MS,
  • Database search and
  • Identified protein annotation- 90 min run.

Per sample

LC120-MS/MS

Peptide sequencing and protein identification by liquid chromatography – tandem mass spectrometry of moderately complex samples. Services include

  • Tandem mass spectrometry analysis using nano-LC/MS/MS,
  • Database search and
  • Identified protein annotation – 120 min run.

Per sample

LC240-MS/MS

Peptide sequencing and protein identification by liquid chromatography – tandem mass spectrometry of very complex samples. Services include

  • Tandem mass spectrometry analysis using nano-LC/MS/MS,
  • Database search and
  • Identified protein annotation – 240 min run.

Per sample

LC-MS/MS-PTM

Peptide sequencing and Post Translational Modification (PTM) Mapping of purified or partially purified proteins by Mass Spectrometry. Services include

  • Tandem mass spectrometry analysis using nano-LC/MS/MS,
  • Database search
  • PTM Mapping and
  • Identified protein annotation.

Per sample

MudPIT Proteomics.

Multi-dimensional Protein Identification Technology and peptide sequencing. Services include

  • Off line SCX fractionation into multiple fractions,
  • Online Reverse Phase (RP) liquid chromatography (LC) – tandem mass spectrometry analysis using nano- LC/MS/MS,
  • Database search and
  • Identified full protein annotation.

Per sample

iTRAQ based Proteomics.

Quantitative proteomics using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ). Labeling with 2-8 tags can be used. iTRAQ Labeling is a non-gel based protein quantitation technique using 5 – 100 µg of protein. 2 to 8 samples can be compared in a single experiment. Services include

  • In-solution digestion
  • iTRAQ labeling
  • SCX cleanup,
  • Tandem mass spectrometry analysis using nano- LC/MS/MS,
  • Database search and
  • Identified full protein annotation.

2-8 samples

iTRAQ-MudPIT Proteomics.

iTRAQ plus MudPIT. Service includes

  • Multi-dimensional offline fractionation of samples into 2-20 fractions,
  • LC-MS/MS analysis of all fractions,
  • Database search and annotation of identified proteins.

Per sample

TMT based Proteomics.

Quantitative proteomics using Tandem Mass Tags (TMT). Labeling with 2-8 tags can be used. TMT Labeling is a non-gel based protein quantitation technique using 5 – 100 µg of protein. 2 to 8 samples can be compared in a single experiment. Services include

  • In-solution digestion
  • TMT labeling
  • SCX cleanup,
  • Tandem mass spectrometry analysis using nano- LC/MS/MS,
  • Database search and
  • Identified full protein annotation.

2-8 sample

TMT-MudPIT Proteomics.

TMT plus MudPIT. Service includes

  • Multi-dimensional offline fractionation of samples into 2-20 fractions,
  • LC-MS/MS analysis of all fractions,
  • Database search and annotation of identified proteins.

Per sample

xMAP analysis

Quantitative protein analysis using Luminex xMAP technology. The level of up to 100 proteins can be measured simultaneously in 50ul of serum, plasma, tissue lysate and other body fluids.

1-100 samples

Metabolomics – PMP

Primary Metabolism Profiling (PMP): This analysis targets carbohydrates and sugar phosphates, amino acids, hydroxyl acids, free fatty acids, purines, pyrimidines, aromatics, exposome-derived chemicals.

Per Sample (minimum 12 samples)

Metabolomics – CLP

Complex Lipids Profiling (CLP): This analysis will target Ceramides, Sphingomyelins, Cholesteryl esters, Oxysterols, Lyso- and phospholipids, mono-, di- and triacylglycerols, galactosyl- and glucuronyllipids.

Per Sample (minimum 12 samples)

Metabolomics – BAP

Biogenic Amines Profiling (BAP): This analysis will target acylcarnitines, TMAO, cholines, betaines, SAM, SAH, nucleotides and nucleosides, methylated and acetylated amines, di- and oligopeptides.

Per Sample (minimum 12 samples)

Metabolomics –TMA

Targeted Metabolite Analyses (TMA): This analysis measures bile acids, steroids, eicososanoids / oxylipins and, very short chain fatty acids using liquid chromatography-QTRAP or gas chromatography-quadrupole mass spectrometry

Per Sample (minimum 12 samples)

Database Search – Protein Identification

Database search for protein identification. The service includes

  • Downloading the most recent database files from NCBI or other relevant sources
  • Database search and
  • Identified protein annotation.

Per search

Database Search – Protein Identification and PTM Mapping

Database search for protein identification and post translational modification mapping. The service includes

  • Downloading the most recent database files from NCBI or other relevant sources
  • Database search and
  • Identified protein annotation.

Per search

Gene Ontology (GO) Annotation

Annotation of proteins using GO classification for biological process, molecular function and cellular component.

Per search

Manual Spectrum Interpretation

Manual Interpretation of mass spectrum

Per hour (Minimum one hour)

 

 

TWO-DIMENSIONAL DIFFERENCE IN-GEL ELECTROPHORESIS (2D-DIGE)

Biostring Inc. utilizes validated sample preparation kits/protocols, robotic spot pickers/digesters, DeCyder difference-in-gel analysis software and advanced mass spectrometers to offer an integrated 2D-DIGE – MS/MS proteomics solution. Proteomic analysis usually starts from sample preparation, to analyses by mass spectrometry (LC-MS/MS) and protein identification and annotation. Data obtained after 2D-DIGE analysis may include protein identity, pI, molecular weight and post translational modifications.

Biostring Inc. has provided 2D-DIGE analysis and proteomics services to NIH, industry, biotechnology companies, pharmaceutical companies, Research Institutes and Universities. Our more than 15 years of experience with 2D-DIGE analysis, proteomics, mass spectrometry and protein expression profiling enables us to offer reproducible picture perfect gels at a competitive price and guaranteed faster turnaround time. We have also developed a variety of complementary 2D-DIGE kits to streamline and standardize protein isolation, protein quantitation, protein qualification and the 2D-DIGE analysis step, thereby reducing the overall time and cost of performing 2D-DIGE analysis.

2D-DIGE offers all the advantages of the classical 2D-PAGE, and most importantly overcomes the inherent disadvantage of gel-to-gel variation and reproducibility problem inherent with the traditional 2D-PAGE approach. 2D-DIGE is presently the most versatile gel-based approach for comparative and semi-quantitative proteomics. Distinct fluorescent tags e.g. Cy 3, 5 and 2 are used to label protein samples and a universal internal standard prior to 1st/2nd dimension electrophoresis. An automated software program is used to detect, quantify and annotate differentially expressed proteins. 2D-DIGE analysis can be applied to protein expression profiling and comparative proteomics by using DeCyder software to identify proteins with statistically-significant difference in expression. This provides unique opportunities for researches in fields including cancer, neuroscience, vision, cardiovascular, Alzheimer, Parkinson, etc, to find important proteins associated with the condition. 2D-DIGE – LC/MS/MS analysis can also be applied to identify post translationally modified proteins (PTM) that are important in cell signaling and protein trafficking.

 

Our 2D-DIGE based comparative proteomics services includes, experimental design, Sample preparation to isolate total proteins using ToPI-DIGE protein isolation Kit, pre-fractionation, quantitation and qualification of proteins, 2D-DIGE analysis, scanning of gels using a Typhoon DIGE-enabled digital scanner, Biological Variation Analysis (BVA) of spots detected to identify differentially expressed proteins with statistically significant difference, robotic spot picking of candidate proteins, robotic in-gel digestion of candidate proteins with trypsin or another enzyme. The enzyme digested proteins are identified by Tandem Mass Spectrometry and annotated.

We offer the following services:

NAME DESCRIPTION UNIT COST
Sample Preparation 1 Homogenization, protein quantitation using ToPA Bradford protein assay kit. Per sample
Sample Preparation 2 Includes, precipitation, buffer exchange, protein quantitation, etc. and the use of ToPI-DIGE, ToPREP and ToPA kits. Per sample
Sample Preparation 3 Depletion of abundant protein e.g. bovine or human serum albumin using ASK-Column kit. Per sample
Sample Preparation 4 Depletion of abundant protein e.g. bovine or human serum albumin using ASK-Solvent kit. Per sample
Sample Preparation 5 Depletion IgY Column Depletion of 14 abundant proteins from serum /plasma using IgY column. Per sample
Sample Preparation 9 Urine protein isolation and concentration. Service includes Isolation, partial purification, and concentration of urine proteins using Urine Protein Isolation kit. Per sample
2D-DIGE Two- Dimensional Difference in-Gel Electrophoresis (2D- DIGE). Quantitative and comparative proteomics using 2D-DIGE. Services include:
  • Experimental design,
  • Sample preparation to isolate total proteins using ToPI- DIGE Protein Isolation Kit,
  • Fractionation, quantitation and qualification of proteins,
  • 2D-DIGE analysis using Cy2, Cy3 and Cy5, scanning of gels using a DIGE- enabled digital scanner and
  • Biological variation analysis of gel images to identify differentially expressed proteins with statistically significant difference.
2 – 10 Samples
Robotic spot picking Selection and picking of fluorescent labels protein spots from 2D-DIGE gels using fully automated ETTAN spot picking robot. 1-96 spots
Robotic spot digestion In gel digestion of protein spots picked from 2D-DIGE gels using a fully automated ETTAN spot digestion robot. 1-96 spots
LC60-MS/MS Peptide sequencing and protein identification by liquid chromatography – tandem mass spectrometry of pure protein samples. Services include:
  • Tandem mass spectrometry analysis using nano-LC/MS/MS,
  • Database search and
  • Identified protein annotation – 60 min run.
Per sample

QUANTITATIVE MULTIPLEXED PROTEIN ANALYSIS USING THE LUMINEX XMAP

We utilize the Luminex xMAP system to provide multiplex protein biomarker monitoring, validation and screening. Multiple target proteins can be monitored simultaneously in the same samples. Kits developed by Luminex strategic partners that have received 510(k) clearance from the FDA can be used on the Luminex system for diagnostic purposes. Custom assays can be developed using antibodies supplied by the client or off the shelf assays can be obtained from strategic partners for most biomedical research applications.

Most biological and clinical samples can be analyzed, including, tissue homogenate, cell lysate, whole blood, serum, plasma, purified proteins and formalin-fixed paraffin embedded samples. Partial list of assays available: cancer markers, cardiac markers, cellular signaling, cytokines, chemokines, and growth factors, endocrine, isotyping, matrix metalloproteinases, metabolic markers, neurobiology, and transcription factors/nuclear receptors.

Why xMAP technology?

  • Multiplexing: Up to 100 bioassays per well; including controls.
  • Parallel analysis: simultaneous quantitation of multiple proteins in the same sample.
  • Sample amount: less than 50ul for up to 100 data points.
  • Reproducibility: provides consistent results and allow for easy quality control.
  • Accuracy: Quantitative results over a 3 to 4 log dynamic range, with strong concordance to ELISA and mass spectrometry.

We offer the following services based on the xMAP technology:

NAME DESCRIPTION UNIT COST
Sample Preparation 1 Homogenization, protein quantitation using ToPA Bradford protein assay kit. Per sample
Sample Preparation 2 Includes precipitation, buffer exchange, protein quantitation, etc. and the use of ToPI-DIGE, ToPREP and ToPA kits. Per sample
Sample Preparation 3 Depletion of abundant protein e.g. bovine or human serum albumin using ASK-Column kit. Per sample
Sample Preparation 4 Depletion of abundant protein e.g. bovine or human serum albumin using ASK-Solvent kit. Per sample
Sample Preparation 5 Depletion IgY Column Depletion of 14 abundant proteins from serum / plasma using IgY column. Per sample
xMAP analysis

Quantitative protein analysis using Luminex xMAP technology. The level of up to 100 proteins can be measured simultaneously in 50ul of serum, plasma, tissue lysate and other body fluids.

1-100 samples
Assay Development for xMAP analysis of Proteins This service involves development assays to target proteins(s) of interest. Per target

QUANTITATIVE MULTIPLEXED RNA ANALYSIS

Biostring Inc. utilizes the Panomics QuantiGene Plex Branched-DNA and Luminex xMAP technologies (QuantiGene-xMAP) to offer multiplexed RNA analysis. Our service includes experimental design, Sample preparation, use of commercially available or in-house developed probes to analyze samples and full statistical data analysis.

Multiple RNA targets can be monitored simultaneously in the same sample. The sensitive and reproducible QuantiGene SinglePlex and MultiPlex technology and Reagent systems has higher sensitivity than most micro arrays and this technology does not require RNA isolation, target amplification by PCR and reverse-transcription.

QuantiGene-xMAP can be applied for research in the following areas: cancer, cardiac disease, cellular signaling, cytokines, chemokines, growth factors, endocrine, matrix metalloproteinases, metabolic disease, neurobiology, transcription factors and nuclear Receptors. Most biological and clinical samples can be analyzed, including, tissue homogenate, cell lysate, whole blood, purified RNA, serum, plasma, and formalin-fixed paraffin embedded samples.

Partial list of assays available: 1L6R, ACTB, BAD, BAK1 (BAK), BCL2, BCL2L1 (BCL-XL), CDKN1A (CDKN1), CFLAR (CFLIP), CSF2, GAPD, IFN-gamma, IL-1 beta, IL-10, IL-2, IL-6, IL-8, NFKB2, NFKBIA (NFKIA), NKFB1, PPIB, Ptk2B (RAFTK), RELA, RELB, TNF, TNFAIP3 (A20), TNFRSF6 (FAS), TNFSF6 (FASL), and VEGF.

Why QuantiGene – xMAP technology?

  • Multiplexing: Up to 100 bioassays per well; including controls.
  • Parallel analysis: Simultaneous quantitation of multiple RNAs in the same sample.
  • Sample amount: Less than 50ul for up to 100 data points.
  • Reproducibility: Provides consistent results and allow for easy quality control.
  • Accuracy: Quantitative results over a 3 to 4 log dynamic range, with strong concordance to micro array and real time PCR.

We offer the following services:

NAME DESCRIPTION UNIT COST
Sample Preparation for QuantiGene -xMAP Preparation of samples for SP1QGX. This service may include homogenization, filtration, fractionation, centrifugation or dilution of samples prior to analysis. Per sample
QuantiGene – xMAP analysis of RNA Quantitative multiplexed RNA analysis using Panomics QuantGene Plex and Luminex xMAP technology. The level of up to 100 RNA targets can be measured simultaneously in 50ul of serum, plasma, tissue lysate and other body fluids. 1-100 samples
Assay Development for QuantiGene – xMAP analysis of RNA This service involves development assays to target RNA(s) of interest. Per target